Mx-1 conditionally immortalized cells

ABSTRACT

This invention relates to methods and compositions of controlling cell distribution within a bioartificial organ by exposing the cells to a treatment that inhibits cell proliferation, promotes cell differentiation, or affects cell attachment to a growth surface within the bioartificial organ. Such treatments include (1) genetically manipulating cells, (2) exposing the cells to a proliferation-inhibiting compound or a differentiation-inducing compound or removing the cells from exposure to a proliferation-stimulating compound or a differentiation-inhibiting compound; exposing the cells to irradiation, and (3) modifying a growth surface of the BAO with ECM molecules, molecules affecting cell proliferation or adhesion, or an inert scaffold, or a combination thereof. These treatments may be used in combination.

This is a division of U.S. application Ser. No. 08/432,698 filed May 9,1995, entitled “Methods and Compositions of Growth Control for CellsEncapsulated Within Bioartificial Organs,” now U.S. Pat. No. 5,843,431,which is a CIP of Ser. No. 08/279,773 field Jul. 20, 1994, now U.S. Pat.No. 5,935,849.

FIELD OF THE INVENTION

This invention relates to methods and compositions for controllinggrowth of cells encapsulated in a bioartificial organ.

BACKGROUND OF THE INVENTION

Bioartificial organs “BAO” are devices which contain living cells andare designed to provide a needed metabolic function to a host.

The cells encapsulated in BAOs supply one or more biologically activemolecules to the host that may be used to prevent or treat many clinicalconditions, deficiencies, and disease states.

For example, BAOs containing insulin secreting cells may be used totreat diabetes. Similarly other diseases such as hypoparathyroidism andanemia may be treated by using cells which secrete parathyroid hormoneand erythropoietin, respectively.

Bioartificial organs may also be used to supply biologically activemolecules for the treatment or prevention of neurodegenerativeconditions such as Huntington's disease, Parkinson's disease,Alzheimer's disease, and Acquired Immune Deficiency Syndrome-relateddementia. Additionally, lymphokines and cytokines may also be suppliedby BAOs to modulate the host immune system. Other biologically activemolecules which may be provided by bioartificial organs include,catecholamines, endorphins, enkephalins, and other opioid or non-opioidpeptides that are useful for treating pain. Enzymatic deficiencies mayalso be treated by using BAOs. Alternatively, the biologically activemolecule may remove or eliminate deleterious molecules from the host.For example, a BAO may contain cells which produce a biologically activemolecule that can be used to “scavenge” cholesterol from a host.

Various “macrocapsule” BAOs are known. See, e.g., Aebischer (U.S. Pat.No. 5,158,881), Dionne et al. (WO 92/03327), Mandel et al. (WO91/00119), Aebischer (WO 93/00128). BAOs also include extravasculardiffusion chambers, intravascular diffusion chambers, intravascularultrafiltration chambers, and microcapsules. See, e.g., Lim et al.,Science 210:908-910 (1980); Sun, A. M., Methods in Enzymology 137:575-579 (1988); Dunleavy et al. (WO 93/03901) and Chick et al. (U.S.Pat. No. 5,002,661).

Because the cells encapsulated in the BAO provide the needed metabolicfunction, it is desirable that those cells optimally supply thebiologically active molecule that effects that function. Typically,differentiated, non-dividing cells may be preferred over dividing cellsfor use in BAOs because they allow for the optimal production of thedesired biologically active molecule. For example, many differentiated,non-dividing cells produce a greater quantity of a desired therapeuticprotein than dividing cells because the expression of differentiationspecific genes and cell division are thought to be antagonisticprocesses. Wollheim, “Establishment and Culture of Insulin-Secreting BCell Lines,” Methods in Enzymology, 192, p. 223-235 (1990). Cellularreplication capacity decreases as cells differentiate. In many cases,proliferation and differentiation are mutually exclusive. Gonos,“Oncogenes in Cellular Immortalisation and Differentiation,” 13,Anticancer Research, p. 1117 (1993).

The use of differentiated tissue is advantageous because the functionalproperties of tissue desired for incorporation into a BAO have mostoften been defined by the properties of differentiated tissue in vivo.Another advantage to the use of differentiated, non-dividing cells isthat the cell number within the BAO will remain relatively constant.This, in turn, leads to more predictable results and stable dosage forthe recipient host. Additionally, differentiated cells are better suitedfor use in BAOs which encapsulate more than one cell type secretingbiologically active molecules. In such BAOs, if dividing cells are used,different cell types may grow at different rates, resulting in theovergrowth of one cell type. By using differentiated, non-dividingcells, the relative proportions of two or more synergistic cell typescan be more readily controlled.

Although in many instances the use of differentiated cells isadvantageous, there have been various problems associated with utilizingdifferentiated cells directly isolated from mammals.

First, there is the potential contamination of the isolated tissue whichmay require that the tissue taken from each animal be subjected tocostly and time-consuming testing to assure that it is pathogen-free.

Second, tissue can be damaged during isolation due to the use ofmechanical or enzymatic isolation procedures in the isolation process.The mechanical manipulations are not always easily standardized,resulting in variability between isolations.

Third, ischemia may occur during isolation causing tissue damage.

Fourth, reproducible yields may be difficult because of variations intissue donors. For example, the age, sex, health, hormonal status of thesource animal can affect the yield and quality of the tissue ofinterest.

Fifth, sometimes there is not enough source tissue to meet the projecteddemand for the BAO. This occurs for example, in a case where the sourcetissue comes from a small sized organ or where the ultimate need-fortissue amounts is high. If the source of the isolated tissue is human,there is frequently a severe shortage of donor tissue.

Sixth, in some cases, it is desirable to genetically modify the cellsused in the BAO. Non-dividing tissue to date has been difficult togenetically modify in vitro and the yields and properties of themodified cells may be uncertain. Thus, because of the foregoingproblems, while the use of differentiated, non-dividing cells isdesirable, a need exists for a method of producing and maintainingdifferentiated, non-dividing cells for encapsulation in BAOs.

Because of these problems, dividing cells and cell lines have beenfavored for use within BAOs to provide the needed biological function.One important advantage in using dividing cells is that such cells maybe grown to large numbers in vitro and screened for pathogens andbanked. This allows an almost unlimited supply of tissue for lowerproduction costs. Selection schemes such as cell sorting or cloning maybe applied to the cell bank to develop subpopulations with improvedcharacteristics. Additionally, dividing cells and cell lines are moreamenable to genetic engineering than differentiated, non-dividing cells.The ability to introduce heterologous recombinant DNA allows many newpossibilities for the alteration of the function or phenotype of cellsto be encapsulated in the BAO. This in turn provides for a greaterdiversity of therapeutic uses for BAOs.

However, as discussed supra, the disadvantages in encapsulatingcontinuously dividing cells in a BAO include poor regulation of cellnumbers in the device that may result in less predictability inproduction of the desired biologically active molecule.

While in most cases it may be desirable to limit or minimize cell growthwithin the BAO, in other cases, e.g., where the BAO is implanted in a“hostile” environment, it may be desirable to allow the cells toproliferate slowly to maintain cell numbers in the BAO.

There is another problem associated with encapsulating cells in general.A variety of cell types have cell adherent properties such that cellstend to adhere to each other and form dense agglomerations oraggregates, especially if there is no adequate substrate available forthe cells. Such cell clusters may develop central necrotic regions dueto the relative inaccessibility of nutrients and oxygen to cellsembedded in the core, or due to the build up of toxic products withinthe core. The necrotic tissue may also release excess cellular proteinswhich unnecessarily flood the host with xeno-proteins or other factorswhich are detrimental to the surviving cells, e.g., factors which elicita macrophage or other immune response. This problem may be exacerbatedwhen cells are encapsulated in a BAO with a semipermeable membranejacket because of diffusional constraints across the membrane. Oftenless oxygen and fewer host supplied nutrients are available within theBAO. In addition, waste products may accumulate in the BAO.

These dense cellular masses can form slowly into dense colonies of cellgrowth or form rapidly, upon the reassociation of freshly-dispersedcells or tissue mediated by cell-surface adhesion proteins. Cells ortissues with a high metabolic activity may be particularly susceptibleto the effects of oxygen or nutrient deprivation, and die shortly afterbecoming embedded in the center of a large cell cluster. Many endocrinetissues, which normally are sustained by dense capillary beds, exhibitthis behavior; islets of Langerhans appear to be particularly sensitivewhen encapsulated.

There is a need to have a method and composition for controlling thegrowth of encapsulated cells which combines the various advantages ofboth proliferating cells and differentiated, non-dividing cells. Thepresent invention provides methods and compositions whereby cells can beproliferated and expanded indefinitely in vitro and where the balancebetween proliferation and differentiation can be controlled when thecells are encapsulated within the BAO so that the device performs in thedesired manner. This invention thus allows regulation of the cell numberwithin the BAO and may therefore provide improved regulation of theoutput level of the capsule. This invention also provides methods forcontrolling the growth of cells by controlling cell location within theBAO, thereby reducing the formation of undesirable necrotic cell coresin the BAO. Controlling the cell number and cell location within the BAOalso provides the advantage of facilitating optimization of the BAOmembrane and other device paramaters to the particular encapsulated celltype. This is because the required device characteristics are morereadily determined for a fixed cell population than for a dividing cellpopulation in the BAO. Additionally, long term delivery of biologicallyactive molecules can be achieved.

SUMMARY OF THE INVENTION

The present invention addresses the foregoing problems by providingmethods and compositions for controlling the distribution of cells (i.e.cell number or cell location in the BAO, or both) when encapsulated in aBAO. The methods and compositions of this invention include (1) methodsand compositions for modification of the cells that are encapsulatedwithin the BAO and (2) methods and compositions for modifying the growthsurfaces within the BAO.

Methods and compositions for cellular manipulation include geneticalteration of the cells with a gene which encodes a product thatinfluences cell proliferation or differentiation. The treatment maycomprise providing a chemical compound or growth factor which inhibitsproliferation or induces differentiation. Alternatively, the treatmentmay comprise removing from the growth medium a chemical compound orgrowth factor which stimulates proliferation or inhibitsdifferentiation. The treatment may be before or after encapsulation inthe BAO, preferably before encapsulation. Additionally, cellproliferation may be controlled by irradiation.

Methods and compositions for growth surface modification include coatingat least one growth surface within the BAO with one or moreextracellular matrix molecules (“ECM”). The ECMs may be coated directlyonto the luminal surface or any inner support within the BAO, or ontomicrosphere carriers (“microcarriers”). Cells or cell-seededmicrocarriers may additionally be suspended in a matrix material thatphysically inhibits cell proliferation. Further, the matrix material maybe derivatized with chemical or peptide derivatives.

In addition, a growth surface of the BAO can be modified by chemicaltreatment to inhibit cell attachment or to enhance cell attachment tothe BAO's luminal surface. Further, the growth surface can be modifiedby addition of an inert scaffold prior to cell loading. The scaffoldphysically inhibits cell outgrowth and provides additional sites forcell attachment. It is to be understood that the various methods andcompositions for cell modification and for growth surface modificationare not mutually exclusive and may be used in combination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the plasmid map of a construct containing a 2.3 kbfragment of the murine Mx1 promoter fused to SV40 early region, followedby a BamH1-Xba1 fragment from mouse beta globin 3′ untranslated region.

FIG. 2 shows NGF secretion (ng/ml/24 h) after 4, 11 and 25 days from BHKcells encapsulated in control, underivatized membranes (shown as “0%” inlegend) or 1% or 5% PEO-PDMS derivatized membranes (shown as “1%” and“5%”, respectively, in legend). Cells were encapsulated with no matrix(shown as “no mat” in legend), a Vitrogen™ matrix (shown as “vit” inlegend), or an agarose matrix (shown as “agse” in legend).

FIG. 3 shows NGF release from BHK cells grown on CultiSphers™ in theabsence of an agarose matrix (legend: n-mat-008, 0709-n-m) or in thepresence of an agarose matrix (legend: agaro-008, agaro-0709).

FIG. 4 shows release of catecholamines from PC12A cells at 1, 14 and 28days after encapsulation in BAOs having a inert PHEMA scaffold. Panel Ashows basal catecholamine release; Panel B shows K⁺-evoked catecholaminerelease. The abbreviations L-dopa, NEPI, epi, DOPAC, DA and HVA in thelegend represent L-dopa, norepinephrine, epinephrine, dopac, dopamine,and homovanillic acid, respectively.

FIG. 5 shows release of catecholamines from PC12A cells at 1, 14 and 28days after encapsulation in BAOs having a inert PHEMA/MMA scaffold.Panel A shows basal catecholamine release; Panel B shows K⁺-evokedcatecholamine release. The abbreviations L-dopa, NEPI, epi, DOPAC, DAand HVA in the legend represent L-dopa, norepinephrine, epinephrine,dopac, dopamine, and homovanillic acid, respectively.

FIG. 6 shows release of L-dopa from SV40/DB4-NGF cells grown onCultisphers™ in the presence of an alginate matrix (legend: CS/AL) or inthe presence of an agarose matrix (legend: CS/AG) at 2, 20, 40 and 80days after encapsulation in BAOs.

DETAILED DESCRIPTION OF THE INVENTION

Definition

As used herein, a “bioartificial organ” or “BAO” is a device which maybe designed for implantation into a host or which may be made tofunction extracorporeally,and either be permanently or removablyattached to a host. A BAO contains cells or living tissues which producea biologically active molecule that has a therapeutic effect on thehost. The BAO, upon implantation in a host recipient, should bebiocompatible. Accordingly, the BAO should not elicit a detrimental hostresponse sufficient to render it inoperable or not therapeuticallyuseful. Such inoperability may occur, for example, by formation of afibrotic structure around the capsule limiting diffusion of nutrients tothe cells therein. Detrimental effects may also include rejection of thecapsule or release of toxic or pyrogenic compounds (e.g. syntheticpolymer by-products) from the BAO to surrounding host tissue.

BAOs comprising encapsulated cells may be constructed withimmunoisolatory properties which hinder elements of the host immunesystem from entering the organ, thereby protecting the cells containedwithin the bioartificial organ from detrimental immune destruction. Theuse of a BAO increases the diversity of cell types that can be employedin therapy. In implanted BAOs, the devices, which may or may not beimmunoisolatory, usually contain the cells or tissues producing aselected product within a semi-permeable physical barrier which willallow diffusion of nutrients, waste materials, and secreted productsinto surrounding host tissue and retain the contained cells, butminimize the deleterious effects of the cellular and molecular effectorsof immunological rejection. Immunoisolatory properties, however, may notbe necessary in all cases (e.g., if the cells are autologous orsyngeneic to the host).

A “biologically active molecule” is one which (a) may function withinthe cell in which it is made or (b) may be expressed on the cell surfaceand affect the cell's interactions with other cells or biologicallyactive molecules (e.g., a neurotransmitter receptor or cell adhesionmolecule), or (c) may be released or secreted from the cell in which itis made and exert its effect on a separate target cell or targetmolecule in the host (e.g., a neurotransmitter, hormone, growth factor,or cytokine).

As used herein, unless otherwise specified, the term “cells” means cellsin any form, including but not limited to cells retained in tissue, cellclusters, and individually isolated cells. The cells used in thisinvention produce at least one biologically active molecule.

Control of cell distribution within the BAO refers to control of thecell number in the BAO, control of the spatial location of cells withinthe BAO, or both.

A wide variety of cells may be used in this invention. These includewell known, publicly available immortalized cell lines as well asdividing primary cell cultures. Examples of publicly available celllines suitable for the practice of this invention include, L-6 cells,MDCK cells, LLC-PK cells, β-CH3 cells, C2 cells, by hamster kidney(BHK), Chinese hamster ovary (CHO), mouse fibroblast (L-M), NIH Swissmouse embryo (NIH/3T3), African green monkey cell lines (includingCOS-a, COS-1, COS-6, COS-7, BSC-1, BSC-40, BMT-10 and Vero), rat adrenalpheochromocytoma (PC12), rat glial tumor cells (C6), RAJI (humanlymphoma) cells, MOPC-31C mouse plasmacytoma cells, MN9D cells, MN9Hcells, ripTAg transgenic mouse derived cells, SCT-1, β-TC cells, Hep-G2cells, AT-T20 cells, beta-cell lines such as NIT cells or RIN cells,Ntera-2 cells (Pleasure et al., Journ. Neuroscience, 12, pp. 1802-15(1992)) and human astrocyte cell lines such as U-373 and U-937.

Primary cells that may be used include, bFGF-responsive neuralstem/progenitor cells derived from the CNS of mammals (Richards et al.,PNAS 89, pp. 8591-8595 (1992); Ray et al., PNAS 90, pp. 3602-3606(1993)), primary fibroblasts, Schwann cells (WO 92/03536), astrocytes,oligodendrocytes and their precursors, myoblasts, and adrenal chromaffincells. For example, one such myoblast cell line is the C₂C₁₂ cell line.

Cells can also be chosen depending on the particular method of growthcontrol and differentiation to be used. For example, stem cells caneasily be used with the methods which induce differentiation byintroducing a chemical substance. Generally, stem cells areundifferentiated cells which in vivo are normally quiescent but arecapable of proliferation and capable of giving rise to more stem cellshaving the ability to generate a large number of progenitor cells thatcan in turn give rise to differentiated or differentiatable daughtercells. Stem cells represent a class of cells which may readily beexpanded in culture, and whose progeny may be terminally differentiatedby the administration of a specific growth factor. See, e.g., Weiss etal. (PCT/CA 92/00283).

Myoblasts are one type of cell that may be encapsulated in a BAOaccording to this invention. Myoblasts are muscle precursor cellsoriginally derived from mesodermal stem cell populations. A number ofmyoblast cell lines are available which can undergo differentiation inculture, e.g., L-6 and β-CH3 cells. Primary myoblasts can be readilyisolated from tissue taken from an autopsy or a biopsy, and can bepurified and expanded. Myoblasts proliferate and fuse together to formdifferentiated, multi-nucleated myotubes. Myotubes no longer divide, butcontinue to produce muscle proteins. While proliferating, myoblasts mayreadily be genetically engineered to produce therapeutic molecules.Methods are known for introducing one or more genes into myoblasts toproduce the desired biologically active molecules. Myoblasts are capableof migrating, fusing into pre-existing fibers, and serving as carriersfor the introduced gene(s). Verma et al. (WO 94/01129); Blau, et al.,TIG, 9, pp. 269-74 (1993); WO 93/03768; WO 90/15863. The engineeredcells may then be encapsulated and allowed to differentiate in the BAOor the differentiated cells may themselves be encapsulated.

The choice of cells also depends upon the intended application. Thecells within the BAO may be chosen for secretion of a neurotransmitter.Such neurotransmitters include dopamine, gamma aminobutyric acid (GABA),serotonin, acetylcholine, noradrenaline, epinephrine, glutamic acid, andother peptide neuro-transmitters. Cells can also be employed whichsynthesize and secrete agonists, analogs, derivatives or fragments ofneurotransmitters which are active, including, for example, cells whichsecrete bromocriptine, a dopamine agonist, and cells which secreteL-dopa, a dopamine precursor.

The cells can be chosen for their secretion of hormones, cytokines,growth factors, trophic factors, angiogenesis factors, antibodies, bloodcoagulation factors, lymphokines, enzymes, and other therapeutic agentsor agonists, precursors, active analogs, or active fragments thereof.These include enkephalins, catecholamines, endorphins, dynorphin,insulin, factor VIII, erythropoietin, Substance P, nerve growth factor(NGF), Glial cell line-derived Neurotrophic Factor (GDNF),platelet-derived growth factor (PDGF), epidermal growth factor (EGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),neurotrophin-4/5, CDF/LIF, bFGF, aFGF, an array of other fibroblastgrowth factors, ciliary neurotrophic factor (CNTF), and interleukins.

It should be understood from the foregoing that the cells useful in themethods of this invention include untransformed cells that secrete thedesired biologically active molecule(s), or cells that can betransformed to do so.

The genes encoding numerous biologically active molecules have beencloned and their nucleotide sequences published. Many of those genes arepublicly available from depositories such as the American Type CultureCollection (ATCC) or various commercial sources. Genes encoding thebiologically active molecules useful in this invention that are notpublicly available may be obtained using standard recombinant DNAmethods such as PCR amplification, genomic and cDNA library screeningwith oligonucleotide probes from any published sequences. Any of theknown genes coding for biologically active molecules may be employed inthe methods of this invention. See, e.g., U.S. Pat. No. 5,049,493; Gageet al., U.S. Pat. No. 5,082,670; and U.S. Pat. No. 5,167,762.

A gene of interest (i.e., a gene that encodes a suitable biologicallyactive molecule) can be inserted into a cloning site of a suitableexpression vector by using standard techniques. These techniques arewell known to those skilled in the art.

The expression vector containing the gene of interest may then be usedto transfect the cell line to be used in the methods of this invention.Standard transfection techniques such as calcium phosphateco-precipitation, DEAE-dextran transfection, lipid-mediated methods, orelectroporation may be utilized.

Methods are provided herein to control the growth of dividing cells,whereby the balance between proliferation and differentiation can becontrolled to provide a supply of differentiated, non-dividingencapsulated cells within the BAO. Methods are also provided to controlthe growth of both dividing and non-dividing cells, whereby celldistribution and cell number within the BAO are controlled, resulting inreduced formation of necrotic cell cores and reduced cellular debris.

Control of Proliferation and Differentiation By Genetic Engineering

Methods and compositions are herein provided for controlling cell growthby genetic alteration of cells with a gene encoding a product thatinfluences cell proliferation or differentiation.

According to one aspect of this invention, conditionally immortalizedcell lines are used to achieve growth control in the BAO. Primary cellsare transformed with a gene encoding a proliferation-promoting product.The proliferation-promoting gene is operatively linked to a regulatablepromoter. The techniques described by Land et al., Nature, 304, pp.596-602 (1983) or Cepko, Neuron, 1, pp. 345-53 (1988) for producingimmortalized cells can be routinely modified to produceconditionally-immortalized cells.

According to this method, cell proliferation (i.e., mitosis) can beinhibited or arrested by decreased expression of aproliferation-promoting gene, such as an oncogene (e.g., c-myc,v-mos,v-Ha-ras, SV40 T-antigen, E1-A from adenoviruses). Reducedexpression of the oncogene is achieved by downregulation, repression orinactivation of the promoter driving oncogene expression when the BAO isimplanted in vivo in a host. Upregulation, activation or derepression ofthe regulatable promoter in vitro results in expression of theproliferation-promoting gene, thereby permitting cell proliferation invitro. Suitable promoters are those which can be downregulated in vivo,including, e.g., glucocorticoid responsive promoters, such as PNMT(Hammang et al., Neuroprotocols, 3, pp. 176-83 (1993) and interferon(“IFN”)-responsive promoters, such as Mx1 (Hug et al., Mol. Cell. Biol.,8, pp. 3065-79 (1988); Arnheiter et al., Cell, 52, pp. 51-61 (1990)),retroviral long terminal repeat promoters, tetracycline responsivepromoters, e.g., the lac promoter, and insulin-responsive promoters. Seealso, McDonnell et al. WO 93/23431. It will be appreciated that choiceof promoter will depend upon the intended implantation site. Thus, e.g.,glucocorticoid or IFN-responsive promoters are useful for implantationin the brain according to this method, since the levels ofglucocorticoid and/or IFN are very low in the brain. Thus, thesepromoters would not be expected to direct significant levels ofexpression of the oncogene upon implantation of the BAO in the brain.

In one embodiment, conditionally-immortalized cells are generated byoperatively linking an oncogene to a regulatable promoter. The promoteris activated or upregulated in the presence of a binding protein.Production of the binding protein can be regulated by operativelylinking the gene encoding the binding protein to a tetracyclineresponsive promoter.

For example, one embodiment contemplates a transformed cell containing aconstitutive promoter driving tet repressor expression. The celladditionally contains a heterologous gene operatively linked to theCMV-IE promoter. If the CMV-IE promoter is flanked with tet operatorsequences, expression from this promoter can be turned off by the tetrepressor. In the presence of tet, transcription occurs because tetbinds with the tet repressor allowing other transcription factors tobind the CMV-IE promoter. According to this embodiment, the oncogene isonly expressed when tetracycline is present. Thus, cells can beproliferated in vitro in the presence of tetracycline.

Several days prior to implantation, tetracycline can be removed toreduce transgene expression, and thus correspondingly reduce or haltcell proliferation in the BAO.

In a specific embodiment using conditionally immortalized cells, growthcontrol is achieved using the Mx1 promoter. The Mx1 gene encodes aprotein which confers resistance to influenza A and B. The Mx1 gene istightly regulated by its promoter. In the absence of interferon (“IFN”),the gene is not expressed and the gene is inducible in the presence ofIFNα and IFNβ. Arnheiter et al., Cell, 52, pp. 51-61 (1990) reported thegeneration of Mx1 transgenic mice that exhibited interferon inducibleexpression of the transgene in several tissues. The SV40 large T-antigenis capable of transforming and immortalizing cells derived from a numberof tissues.

In one embodiment, the mouse Mx1 promoter can be fused with the SV40early region and the chimeric gene used to generate transgenic mice. Thetight regulation afforded by the Mx1 promoter elements allows one tocontrol oncogene expression in tissues or in cell cultures prepared fromthe transgenic animals, thereby allowing creation ofconditionally-immortalized cell lines.

In the presence of IFNα or IFNβ, the cell lines produced in this mannercan be expanded arithmetically as with most other cell lines. Celldivision can be halted by removal of IFNα or IFNβ, either before orafter encapsulation. In a preferred embodiment, neural stem cells(neurospheres) can be prepared from transgenic mice containing theMx1-SV40 T-antigen construct using the method of Weiss (PCT/CA92/00283). The conditionally immortalized neural stem cell line soobtained can then be encapsulated and implanted in vivo in a host.

Additionally, if desired, the conditionally immortalized neural stemcell line can be further genetically modified to release any of a numberof growth factors or neurotransmitter molecules, according to standardtechniques. Other IFN-responsive promoters may also be useful in thisembodiment. These promoters include metallothionein, H-2K^(b), H-2D^(d),H-2L^(d), HLA-A3, HLA-DRα, an HLA class I gene, 202, 56K, 6-16, IP-10,ISG15, ISG54, and 2′,5′-oligo(A) synthetase. See, Hug et al., Mol. Cell.Biol., 8, pp. 3065-79 (1988).

This embodiment is particularly suited for cells to be encapsulated inBAOs for implantation in the brain. Circulating levels of IFNα and IFNβin the brain are sufficiently low that transcriptional activity drivenby the Mx1 promoter is insufficient to result in cell proliferation. Inthe founder transgenic animals, the expression of T-antigen could beinduced in several tissues, but the natural expression of the oncogenewas seen only in the thymus. However, thymic expression of the oncogeneis a relatively common phenomenon in transgenic animals expressing theSV40 early region. Thus, in the absence of significant oncogeneexpression, the cells can be kept in a near quiescent state in vivo.

Another embodiment makes use of the observation that in traditionalretroviral infection techniques to genetically engineer cells for use invivo, retroviral promoters, e.g., the long terminal repeat (“LTR”)promoter, are used. See, e.g., Gage et al. (U.S. Pat. No. 5,082,670).The expression of genes driven by these promoters is typicallydownregulated in vivo. It is thought that this downregulation ismediated by circulating cytokines. This invention makes-use of thisnormally detrimental downregulation of retroviral genes to stop ordecrease cellular proliferation when cells are encapsulated within theBAO and implanted in vivo. In this instance, an immortalizing gene(oncogene) is driven from the LTR. This gene will “immortalize” thecells while they are maintained and expanded in vitro. Followingimplantation, in the presence of cytokines, the “immortalizing” oncogeneis downregulated, proliferation decreases or stops and the cells maybecome quiescent within the device.

According to this embodiment conditionally-immortalized cells may beproduced by retroviral infection or DNA transfection with cDNAcontaining an oncogene (e.g. c-myc, v-mos, v-Ha-ras, SV40 T-antigen,E1-A from adenoviruses) operatively linked to a retroviral promoter,e.g., the LTR promoter. We prefer Moloney murine leukemia virus (MLV),Rous sarcoma virus (RSV), and mouse mammary tumor virus (MMTV) promotersequences.

These transformed cells will normally express the oncogene in vitro.Successfully transformed cells will be grown in culture usingestablished culture techniques. LTR-transgene expression can bestimulated by the addition of dexamethasone or epidermal growth factorto shorten the amount of time needed to culture the transformed cells.By exposing the cells to cytokines, e.g., gamma-interferon (IFN-γ),TNF-α and transforming growth factor-β (TGFβ), preferably several daysprior to encapsulation and implantation, mitosis can be reduced byhindering LTR-driven transgene expression. Schinstine and Gage,Molecular and Cellular Approaches to the Treatment of NeurologicalDisease, 71, ed. Waxman, S. G. (1993); Seliger et al., J. Immunol., 141,pp. 2138-44 (1988); Seliger et al., J. Virology, 61, pp. 2567-72 (1987);Seliger et al., J. Virology, 62, pp. 619-21 (1988).

Any suitable cell can be conditionally immortalized according to theabove methods. One of ordinary skill in the art can determine thesuitability of a given cell type for conditional immmortalization byscreening methods well known in the art, including according to themethods provided herein.

Methods are provided herein for growth control of immortalized celllines or other continuously proliferating cells by transforming thesecells to include tumor suppressor genes, e.g., the p53 gene or RB gene,to halt or reduce proliferation. Tumor suppressor genes, oranti-oncogenes, are believed to be growth-constraining genes. See, e.g.,Weinberg, Neuron, 11, pp. 191-96 (1993). For example, a wild-typep53-activated fragment 1 (WAF1) can suppress tumor cell growth inculture. It is theorized that genes induced by the p53 protein maymediate its biological role as a tumor suppressor. El-Deiry et al.,“WAF1, a Potential Mediator of p53 Tumor Suppression,” Cell, 75, pp.817-825 (1993). The WAF1 gene is also referred to as the CIP1 gene.Other p53-mediated growth arresting genes include GADD45 and GADD153 (orCHOP). See Ron Proc. Natl. Acad. Sci. USA, 91, pp. 1985-86 (1994). Thestandard techniques for transforming cells with heterologous DNAdiscussed above can be used here.

According to one embodiment, immortalized cells or continuouslyproliferating cells are transformed with a tumor suppressor geneoperatively linked to a regulatable promoter. Use of a suitableregulatable or inducible promoter allows expression of the transgene tobe downregulated or “turned off” when the transformed cells are culturedin vitro, thus permitting expansion. Upon encapsulation andimplantation, the promoter is “induced,” or upregulated, and expressionof the tumor suppressor gene occurs, resulting in reduced or halted cellproliferation.

The tyrosine hydroxylase and erythropoeitin promoters may be useful inthis aspect of the invention. These promoters are typically“downregulated” under high O₂ conditions, such as those encountered invitro, but are “upregulated” under low O₂ conditions, like those thatcells encounter upon encapsulation in a BAO and implantation in a host.

In addition, suitable coupled or derepressible promoter systems may beused to achieve the desired regulation of the proliferation-suppressinggene. One suitable system, e.g., involves use of the AP1 promoter andthe lac operator/PGK1 promoter system described by Hannan et al., Gene,130, pp. 233-39 (1993). The AP1 promoter is operatively linked to thelac repressor gene. The lacO (lac operator) and 3-phosphoglyceratekinase (PGK1) promoter is operatively linked to theproliferation-suppressing gene. Addition of exogenous phorbol ester invitro induces the AP1 promoter, resulting in expression of the lacrepressor protein. In the presence of repressor protein, the lacO-PGK1promoter construct is repressed, and no expression of theproliferation-suppressing gene occurs. In the absence of phorbol esterin vivo, no repressor protein is expressed, the lacO-PGK1 promoter isderepressed, and the proliferation-suppressing gene is expressed.

According to one method, a suitable cell is transformed with a geneencoding a differentiation-inducing product. Thisdifferentiation-inducing gene is operatively linked to a regulatablepromoter. According to this method, the differentiation-inducing genewould be expressed upon encapsulation and in vivo implantation in ahost. However, expression can be arrested or inhibited in vitro byappropriate downregulation, repression or inactivation of theregulatable promoter, thus allowing expansion of a desired cell or cellline in vitro. This method can be used with dividing cells, or primarycells that have been immortalized. High mobility group chromosomalprotein 14, “HMG,” is one example of a gene involved in regulatingdifferentiation of cells. Any suitable promoter that is upregulated invivo but which can be “turned off” or downregulated in vitro can be usedin this embodiment, as discussed supra for use withproliferation-arresting genes. In addition, any suitable derepressiblepromoter system can be used, as discussed supra, for the regulation oftumor suppressor gene expression.

Another method of growth control uses antisense RNA or DNA, or theirderivatives. Antisense RNA or DNA is a single-stranded nucleic acidwhich is complementary to the coding strand of a gene or to the “coding”mRNA produced from transcription of that gene. If the antisense RNA ispresent in the cell at the same time as the mRNA, the antisense RNAhybridizes to the mRNA forming a double strand which then cannot betranslated by ribosomes to make protein. Antisense RNA can beadministered to cells either via microinjection or bulk addition toculture medium. The preferred method of the instant invention is totransfect target cells with eukaryotic expression vectors. Neckers etal., “Antisense Technology: Biological Utility And PracticalConsiderations”, Am. J. Physiol., 265 (Lung Cell. Mol. Physiol., 9), pp.L1-L12 (1993).

According to this embodiment, an antisense gene encoding antisense RNAto either a proliferation-inducing gene or a tumor suppressor gene canbe operatively linked to an inducible promoter. When the promoter isinduced, antisense RNA is produced. If the transformed cells contain aproliferation-inducing gene, according to this embodiment, antisense RNAproduction would be halted or downregulated in vitro to allow for cellexpansion, and upregulated in vivo, to achieve cessation or reduction ofproliferation.

Alternatively, if the transformed cells contain a tumor suppressor gene,antisense RNA production would be upregulated in vitro and downregulatedin vivo to achieve the desired growth control.

In addition, antisense technology could be used to construct anyantisense gene to a gene encoding a product essential for proliferationor differentiation. Appropriate induction of the expression of theantisense gene would allow one of skill in the art to achieve thedesired growth control of encapsulated cells according to thisinvention.

It is preferred to use a regulatable promoter/gene construct that can bemanipulated in vivo in the event that it becomes necessary or desirableto induce further cell proliferation in vivo. For example, in theMx1/SV40 construct discussed supra, IFN can be added locally orsystemically to induce oncogene expression. An increase in cell divisionin vivo in the BAO may be desirable to increase cell number to replacedead cells in the BAO, or to achieve increased output of the desiredbiologically active molecule from the BAO.

Control of Growth and Differentiation by Use of Chemical Compounds

According to another method of this invention, cells may be exposed to atreatment which inhibits proliferation or induces differentiation. Insome methods, the treatment comprises providing a chemical compound orgrowth factor. In other methods, the treatment comprises removing achemical compound or growth factor from the growth medium. The treatmentmay be before or after encapsulation in the BAO, preferably beforeencapsulation.

The protein or chemical compound used depends on the cell type and thedesired effect. One of ordinary skill in the art could screen a givencell type for its responsiveness to a selected compound or protein, withroutine techniques.

In one method, cell distribution is controlled by a treatment thatcomprises removing a proliferation-inducing chemical compound or growthfactor from the cell growth medium. In one embodiment, growth factors,such as epidermal growth factor (“EGF”), transforming growth factor a(“TGF-α”), amphiregulin, or any other suitable agent, can be used toinduce proliferation of stem or progenitor cells, including cells fromembryonic sympathetic ganglia and immortalized progenitor cells,preferably neural stem cells (Weiss, PCT/CA 92/00283). This allowsmaintenance and expansion of a supply of neuronal precursor cells invitro. When encapsulated in the absence of these proliferation-inducinggrowth factors, the neuronal precursor cells cease dividing anddifferentiate.

The neuronal precursor cells may be further induced to differentiate bytreatment with, e.g., phorbol ester, or growth on a fixed substrate,including ionically charged surfaces such as poly-L-lysine andpoly-L-ornithine and the like. Differentiation may also be induced bytreatment with a member of the FGF family in combination with at least 1member of either the ciliary neurotrophic factor (CNTF) or nerve growthfactor (NGF) family of factors as described in Ip et al. (WO 94/03199).

In another embodiment, a multilineage growth factor produced in thestroma, also termed “mast cell growth factor,” “stem cell factor,”“c-kit-ligand,” or “Steel factor,” can be used to induce proliferationof hematopoietic stem cells. To maintain a supply of dividing cells invitro, hematopoietic stem cells are cultured in the presence of mastcell growth factor. To arrest or reduce proliferation, the mast cellgrowth factor is removed from the culture medium. This can be donebefore or after encapsulation, preferably before encapsulation.

Examples of other multilineage growth factors that promote proliferationinclude interleukin-3 and granulocyte-macrophage colony-stimulatingfactor. Mast cell growth factor can also affect cell growth incombination with other multilineage growth factors, or lineage specificgrowth factors, e.g., erythropoietin. For example, mast cell growthfactor is thought to act synergistically with IL-3 in inducingproliferation and differentiation of highly enriched murinehematopoietic stem cells. Galli et al., “The Biology of Stem CellFactor, a New Hematopoietic Growth Factor Involved in Stem CellRegulation,” Int. J. Clin. Lab. Res., 23, pp. 70-77 (1993).

In another method of this invention, control of cell distribution in theBAO may be achieved by providing a chemical compound or growth factorwhich inhibits cell proliferation or induces differentiation. Anysuitable proliferation-inhibiting or differentiation-inducing compoundmay be used according to this method.

It will be appreciated that different cell types may respond differentlyto various chemical compounds. One of ordinary skill in the art canroutinely screen a particular compound to determine its effectiveness inaffecting proliferation or differentiation of a given cell type.

In one embodiment, cytokines, including, e.g., transforming growthfactor β1 (TGFβ1), may be used to arrest or inhibit cell proliferationor to induce cell differentiation. For example, decreased proliferationand enhanced differentiation in BHK cells can be achieved by exposure toTGFβ1 and ascorbate. Similarly, TGFβ1 can be used to inducedifferentiation in fibroblast cells and also as a growth inhibitor ofkeratinocytes and endothelial cells. Phillips et al., “Ascorbic Acid andTransforming Growth Factor-β1 Increase Collagen Biosynthesis viaDifferent Mechanisms: Coordinate Regulation of Proα1(I) and Proα1(III)Collagens,” Archives of Biochemistry and Biophysics, 295, pp. 397-403(1992).

In another embodiment, TGFβ1, serotonin, or FGF may be used to controlthe growth of neuroendocrine cells. The growth of neuroendocrine cellscan be regulated by their own products in an autocrine fashion. TGFβ1 isan autocrine growth-inhibitory factor for human pancreatic carcinoidcells (BON), while FGF and serotonin are autocrine growth-stimulatoryfactors. The inhibitory effect of TGFβ1 on the growth of BON cells canbe reversed by addition of serotonin. Townsend Jr. et al., “Studies ofGrowth Regulation in a Neuroendocrine Cell Line,” Acta Oncologica, 32,pp. 125-130 (1993).

A variety of other chemicals may also be used according to the methodsof this invention to arrest or inhibit proliferation or inducedifferentiation of cells. These chemicals include mitomycin C,5-bromo-deoxyuridine (BrdU), prostaglandin E₁ (PGE₁), dibutyryl cAMP,1-β-D-arabinofuranosyl cytosine (Ara-C), nicotinamide, and heparin.Mitomycin may be particularly suited for controlling proliferation ofencapsulated βHC cell lines. See, e.g., Radvanyi et al., Mol. Cell.Biol., 13, pp. 4223-27 (1993).

Sometimes a combination of chemicals can be used. Human neuroblastomacells IMR-32 may be induced to differentiate in vitro when treated withmitomycin C and BrdU or PGE₁ and dibutyryl cAMP (dbcAMP). Gash et al.,“Amitotic Neuroblastoma Cells Used for Neural Implants in Monkeys,”Science, 233, pp. 1420-22 (1986). Serial pretreatments of humanembryonal rhabdomyosarcoma cell line with Ara-C results in marked growthinhibition in vitro, loss of tumorigenicity in vivo, and a moredifferentiated phenotype even following removal of the compound. Crouchet al., “Ara-C Treatment Leads to Differentiation and Reverses theTransformed Phenotype in a Human Rhabdomyosarcoma Cell Line,”Experimental Cell Research, 204, pp. 210-16 (1993). Nicotinamide (NIC)is thought to induce differentiation and maturation of human fetalpancreatic islet cells. Otonkoski et al., “Nicotinamide Is a PotentInducer of Endocrine Differentiation in Cultured Human Fetal PancreaticCells,” J. Clin. Invest., 92, pp. 1459-66 (1993).

The addition of dbcAMP has also been reported to influence thedifferentiation of developing tissues. For example, dbcAMP is thought tomodulate the differentiation of astrocyte precursors, induce neuriteformation in PC12 cells, and stimulate Schwann cell proliferation.Baron-Van Evercooren et al., “Schwann Cell Differentiation in vitro:Extracellular Matrix Deposition and Interaction,” Dev. Neurosci., 8, pp.182-96 (1986). Similarly, differentiation of Schwann cells can beinduced by exposure to ascorbate. Ibid.

Further, sialoglycopeptide (“SGP”) molecules may be used to inhibit orarrest cell proliferation. For example, an 18 kDa cell surfacesialoglycopeptide isolated from intact bovine cerebral cortex cellsarrested proliferation of exponentially growing Swiss 3T3 cells. See,e.g., Toole-Simms et al., Jour. Cell. Physiol., 147, pp. 292-97 (1991);Fattaey et al., Exp. Cell. Res., 194, pp. 62-68 (1991). Numeroustransformed and untransformed cell types have been shown to be sensitiveto some SGPs. These cells include epithelial-like and fibroblast cellsfrom a broad spectrum of vertebrate and invertebrate species. See, e.g.,Fattaey et al., Jour. Cell. Physiol., 139, pp. 269-74 (1989)incorporated herein by reference.

It will be appreciated that some of the foregoing treatments may onlyhave a transient effect on proliferation and differentiation. In suchcases it may be desireable to provide a continuously replenished supplyof the compound or growth factor to the encapsulated cell when implantedin vivo in the host. This can be accomplished by use of a bioerodablepolymer non-cellular source of the growth factor or compound, or byco-encapsulating a cellular source of he growth factor or compound, orany other suitable means. See, e.g., U.S. Pat. No. 5,106,627 and5,156,844.

Control of Growth By Irradiation

Cell proliferation can also be controlled through exposure of cells to asuitable dose of irradiation, e.g., x-rays, ultraviolet (UV) radiation,and the like. When cells are subjected to irradiation, their progressionthrough the cell cycle may be arrested. The critical dose rate, orminimum dose rate can be determined for a chosen cell type using methodsknown in the art. See, e.g., Stanley and Lee, Radiat. Res., 133, pp.163-9 (1993); Mitchell et al., Radiat. Res., 79, pp. 537-51 (1979). Forexample, normal human epidermal keratinocytes irradiated with 5 and 10mJ/cm² ultraviolet B(UVB) radiation showed a significant (up to 78%)decrease in proliferation 3 to 5 days post-irradiation. Prystowsky etal., J. Invest. Dermatol., 101, pp. 54-58 (1993). Yi et al., RadiationResearch, 133, pp. 163-69 (1993) provide a method for calculating thelowest dosage required to stop cell proliferation by exposure to x-rays.

Control of Growth and Differentiation By Use of Extracellular MatrixMolecules

Methods are provided herein for the control of cell distribution in aBAO by modification of a growth surface with a growth controllingextracellular matrix (“ECM”) (or components thereof) alone or incombination with a growth controlling physical matrix or other growthregulating substances.

In living tissue, the ECM is formed from a variety of proteins andpolysaccharides which are secreted by cells and assembled into a networkin proximity to the cells that secreted them. ECM molecules includeglycosaminoglycans and proteoglycans, such as chrondroitin sulfate,fibronectin, heparin sulfate, hyaluron, dermatan sulfate, keratinsulfate, laminin, collagen, heparan sulfate proteoglycan (HSPG) andelastin. In particular, collagen is a major component of ECM in vivo.ECM molecules are known to cause decreased cell proliferation andincreased cell differentiation. In addition, acellular ECM when used inthe methods of this invention may influence the spatial location ofcells encapsulated in the BAO.

ECM may be obtained by culturing cells known to deposit ECM, includingcells of mesenchymal or astrocyte origin. Schwann cells can be inducedto synthesize ECM when treated with ascorbate and cAMP. These ECMcomponents resemble a precursor form of the basement membrane whichsupport Schwann cell proliferation. Furthermore, naturally produced ECMfrom endothelial cells and a reconstituted basement membrane gel fromEngelbreth Holm-Swarm tumor cells (EHS) supports the growth anddifferentiation of various epithelial and endothelial cells. Baron-VanEvercooren et al., “Schwann Cell Differentiation in vitro: ExtracellularMatrix Deposition and Interaction,” Dev. Neurosci., 8, pp. 182-96(1986).

In one embodiment, growth control is achieved by coating a growthsurface in the BAO with ECM (or its growth controlling components). Weprefer seeding the growth surface in the BAO with cells that produceECM, and culturing the cells until confluent. The cells are then treatedwith detergent and NH₄OH. The resulting BAO, with acellular ECM coatedon a growth surface, is then used to encapsulate cells that produce thedesired biologically active molecule.

In another embodiment, ECM is prepared substantially in the same mannerin vitro, lyophilized, fragmented and mixed with cells as a suspension.The cell/ECM fragments are then co-loaded into the BAO.

Cells grown in presence of some ECM molecules show decreasedproliferation and increased differentiation compared to cells grown inconventional monolayer culture. For example, adrenocortical cells, knownto synthesize certain steroid hormones such as aldosterone, exhibitdecreased proliferation when grown in vitro in the presence of collagengel. Fujiyama et al., “Influence of Extracellular Matrix on theProliferation and Differentiation of Adrenocortical Cells in Culture,”Path. Res. Pract., 189, pp. 12051-14 (1993).

Schwann cells may also exhibit decreased proliferation and increaseddifferentiation when cultured in the presence of collagen.

Endocrine cells are also known to differentiate in vitro when grown onsurfaces coated with a combination of type IV collagen and HSPG. Type IVcollagen is necessary for cell adhesion and the HSPG inducesdifferentiation. de Bruine et al., “Extracellular Matrix ComponentsInduce Endocrine Differentiation In Vitro in NCI-H716 Cells,” AmericanJournal of Pathology, 142, pp. 773-782 (1993).

Various growth factors or chemical compounds, including those discussedsupra, may be added to the ECM components to further control the growthand differentiation of cells. Growth factors may be administered to thecells in vitro prior to implantation or to the cells in vivo, or both.See, e.g., U.S. Pat. Nos. 5,156,844 and 5,106,627, which refer tomethods for delivering growth factors using either a co-encapsulatedcellular or non-cellular source of the growth factor. In addition, theECM molecules may be derivatized with growth controlling peptidesaccording to known techniques.

For example, transforming growth factor-β, which modulates cell growthon its own, and which reversibly binds to certain ECM molecules (e.g.decorin), can be added to ECM to potentiate the growth-inhibitingeffects of ECM molecules.

Likewise, heparin has also been shown to prevent the growth of bothuntransformed cells and transformed cell lines. Matuoka et al., CellStructure and Function, 9, p. 357 (1984).

Basic fibroblast growth factor (bFGF) has also been reported to enhanceendocrine cell differentiation when added along with ECM components.See, de Bruine et al., “Extracellular Matrix Components Induce EndocrineDifferentiation In Vitro in NCI-H716 Cells,” American Journal ofPathology, 142, pp. 773-782 (1993).

Growth factors may exhibit different effects on cells when combined withdifferent components of ECM. For example, fibroblast growth factor (FGF)has been shown to be an effective differentiating factor and a weakmitogen for chromaffin cells grown on laminin. However, when FGF isadded to chromaffin cells grown on collagen, FGF is a weakdifferentiation factor and a strong mitogen. This behavior has also beenshown for the cyclic AMP analogue 8-(4-chlorophenylthio) cyclic AMP. Chuet al., Neuroscience, 95, pp. 43-54 (1994).

Table 1 is a partial list of ECM molecules growth factors and chemicalcompounds known to influence proliferation and differentiation inparticular cell types.

TABLE 1 ECM MOLECULES, GROWTH FACTORS AND CHEMICAL COM- POUNDSINFLUENCING PROLIFERATION OR DIFFERENTIATION Differentiation Inducer/Cell Type Growth Inhibitor Proliferation Promoter Schwann ascorbate;collagen TGF-β; dbcAMP (Vitrogen ™); Cultisphers/ agarose PC12 NGF;dbcAMP; SGP Fibroblasts TGF-β-1; Cultisphers/ Vitrogen ™ agarose;ascorbate; SGP Myoblasts collagen; ascorbate Neural stem laminin;Peptite 2000; EGF; bFGF; TGF-α; Cultisphers/Peptite 2000; amphiregulinphorbol ester; heparin; FGF and (CNTF or NGF) Human embryonal Ara-Crhabdomyosarcoma cell line Human fetal Nicotinamide (NIC) pancreaticislet cells Astroblasts dbcAMP Swiss 3T3 SGP Adrenocortical CollagenEndocrine Type IV Collagen + HSPG; bFGF + ECM components ChromaffinFGF + laminin; 8-(4- FGF + collagen; 8-(4- chlorophenylthio)cyclicchlorophenylthio) AMP + laminin cyclic AMP + collagen Hematopoietic stemMast cell Growth cells Factor BHK TGFβ-1 + Ascorbate; ECM from E15 ratmeningeal cells Keratinocytcs TGFβ-1 Endothelial cells TGFβ-1Neuroendocrine TGFβ-1 TGFβ-1 + Ascorbate; (human pancreatic Serotonin;FGF caranoid cells (BON)) Human neuro- Mitomycin C + BrdU; blastoma Cellline PGE₁ + dbcAMP; SGP IMR-32 SCT-1 Collagen; Ascorbate

The growth surfaces within the BAO include the luminal surfaces of theBAO, and additionally include other growth surfaces, such as an innersupport, that may be encapsulated within the BAO.

Microcarriers may provide a surface for cell growth. Use ofmicrocarriers can allow a greater number of cells to be encapsulated andevenly distributed within the BAO, especially for cells that becomegrowth contact inhibited. Several types of microcarriers arecommercially available, including Cytodex (Sigma, St. Louis, Mo.)dextran microcarriers, and CultiSpher™ (HyClone Labs, Logan, Utah)macroporous gelatin microcarriers and glass microcarriers. Thesemicrocarriers are often used for the culture of anchorage dependentcells. Cell lines which have been shown to grow on macroporous gelatinmicrocarriers include OBHK, BHK-21, L-929, CHO-K1, rCHO, MDCK, V79, F9,HeLa, and MDBK. Microcarriers may also be made of or coated with otherECM molecules (such as FACT™ collagen coated microcarriers (Solo HillLabs, Ann Arbor, Mich.)), or acellular ECM, substantially as describedabove.

In one preferred embodiment cells producing the desired biologicallyactive molecules can be seeded onto the ECM coated microcarrier surfacesand cultured on the microcarriers in vitro, prior to encapsulation andimplantation. Cherksey (WO 93/14790) refers to the culturing of cells onglass or plastic microbeads and subsequent implantation of themicrobeads into the brain of a recipient.

In another embodiment according to this invention, cells seeded onmicrocarriers may be suspended in the presence of a suitablegrowth-inhibiting matrix and then encapsulated in the BAO. Such matrixmaterial (e.g., agarose or agar for fibroblasts; collagen foradrenocortical cells) physically inhibits further cell outgrowth. Suchhydrogel matrices are described in, e.g., Dionne WO 92/19195,incorporated herein by reference.

According to another aspect of this invention, agarose may also be usedas a substitute for ECM by derivatization with peptide sequences toaffect cell attachment to the matrix. For example, agarose hydrogels maybe derivatized with peptide sequences of laminin or fibronectin.

In this method, cells are suspended in 3-D matrices composed of agarosederivatized with a peptide sequence that recognizes a cell surfacereceptor molecule involved in cell adhesion. Several peptide sequenceshave been shown (in 2-D) to promote cell adhesion. See, e.g.,Pierschbacher et al., Science, 309, pp. 30-33 (1984); Graf et al.,Biochemistry, 26, pp. 6896-900 (1987); Smallheiser et al., Dev. BrainRes., 12, pp. 136-40 (1984); Jucker et al., J. Neurosci. Res., 28, pp.507-17 (1991). The derivatized agarose matrices of this invention allowpresentation of the appropriate molecular cues for cell adhesion in 3-D.The agarose concentration is preferably 1.25% w/v or less, mostpreferably about 1.0%. We prefer RGD-containing sequences (i.e.ArgGlyAsp; AA₂-AA₄ of SEQ ID NO:2), YIGSR-containing sequences(TyrIleGlySerArg; AA₅-AA₉ of SEQ ID NO:1), IKVAV-containing sequences(IleLysvalAlaVal; AA₁₁-AA₁₅ of SEQ ID NO:3), and the like.Derivatization can be achieved using a bi-functional coupling agent,such as 1′1, carbonyldiimidazole or any other suitable method.

One particular advantage of using agarose instead of ECM components isthat naturally occurring ECM components may be enzymatically degradedover time in vivo while agarose is not as readily degraded. The use ofagarose is also advantageous because it is a defined product unlikematerials like Matrigel®, which is derived from a tumor cell line andtherefore an undefined mixture. Specifically, it has been shown thatMatrigel contains bFGF, a potent mitogen for many cell types. Agarose isa clear, thermoreversible hydrogel made of polysaccharides. In additionto physically restricting cell outgrowth, agarose itself may inhibitproliferation and induce differentiation. See, e.g., Aulthouse, in“Expression of the Human Chondrocyte Phenotype In Vitro,” In VitroCellular & Developmental Biology, 25, pp. 659-668 (1989).

Agarose can be chemically modified by derivatives, e.g., PEO-PDMS, tofurther inhibit cell outgrowth, preferably without toxic effects to thecells.

It will be appreciated that different cell types may exhibit differentresponsiveness to a given ECM molecule, or to acellular ECM from aparticular source. See, e.g., End and Engel, “Multidomain Proteins OfThe Extracellular Matrix And Cellular Growth”, pp. 79-129, in ReceptorsFor Extracellular Matrix, [Eds] McDonald and Mecham, Academic Pree, NewYork (1991), herein incorporated by reference. One of ordinary skill canreadily screen a cell type to determine its responsiveness to an ECMmolecule or to acellular ECM from a specific source, to determine itseffectiveness in controlling cell distribution.

Growth Control by Growth Surface Modification in the BAO

Methods are provided herein for cell growth control in a BAO bychemically modifying growth surfaces to control cell number and celllocation within the BAO. Growth surfaces within the bioartificial organcan be modified to control cell attachment to the growth surface. Thegrowth surface within the BAO can be the luminal surface of the BAO, oran internal membrane, microcarrier or inner support placed inside theBAO. With the microcarrier and inner support embodiments, cells can becultured on these structures in vitro and subsequently encapsulated inthe BAO for implantation.

The BAO membrane may be modified by a number of different known methods,including chemical modification, to produce carboxylic acid groups,amine groups, or hydroxyl groups or other reactive functional groups, orit can be modified by absorption. These reactive functional groups,otherwise not present on the polymer backbone, can subsequently be usedas sites for further derivatization.

In one embodiment, the luminal surface of the BAO is modified to promotecellular attachment thereto. Controlled cell attachment to the luminalsurface may be useful in enhancing cell survival. By attaching the cellspreferentially to the membrane, an even distribution of cells inside thecapsule can be achieved with fewer cells than that are used inimmobilization techniques using a hydrogel suspension. The use of fewercells results in a lesser amount of cellular debris. Another benefit isthe enhanced diffusion of nutrients to the cells because the cells arein close contact with the membrane. If the membrane modification is usedwithout a matrix material within the capsule, complications of transportthrough the gel and adsorption of proteins or cell products to thematrix material can also be avoided. Cellular attachment may be promotedby treatment of the BAO luminal surface with poly(d-lysine) of variousmolecular weights. The poly(d-lysine) can be adsorbed onto the BAOluminal surface from a pH 11 buffered solution. We prefer poly(d-lysine)of about 67,000 g/mole.

In addition, peptide derivatives, e.g., RGD containing sequences(ArgGlyAsp; AA₂-AA₄ of SEQ ID NO:2), YIGSR-containing sequences(TyrIleGlySerArg; AA₅-AA₉ of SEQ ID NO:1), including CDPGYIGSR(CysAspProGlyTyrIleGlySerArg; SEQ ID NO:1), as well as IKVAV containingsequences (IleLysValAlaVal; AA₁₁-AA₁₅ of SEQ ID NO:3) (preferablyCysSerArgAlaArgLysGlnAlaAla SerIleLysValAlaValSerAlaAspArg (SEQ IDNO:3)), have been found to be particularly useful in promoting cellularattachment. For example, RGD (ArgGlyAsp; AA₂-AA₄ of SEQ ID NO:2), themost common of these peptides can be chemically attached to the BAOmembrane, using known techniques. Some RGD (ArgGlyAsp; AA₂-AA₄ of SEQ IDNO:2) containing molecules are commercially available—e.g.,PepTite-2000™ (Telios).

In another embodiment, the BAO membrane can be modified to inhibit cellattachment through adsorption of, e.g., PEO-PDMS orpoly(d-lysine)-alginate. We prefer PEO-PDMS modification, particularlyif the growth surface is porous. This is because PEO-PDMS will tend todiffuse through the pores and adsorb to the surface as it passes throughthe pores through hydrophobic-hydrophobic bonding. In particular, lowmolecular weight (600-3000 g/mole) PEO-PDMS is preferred.

This embodiment is particularly useful when cells are grown onmicrocarriers and encapsulated in the BAO. In this manner, an even celldistribution may be achieved, cell number may be controlled, and celladhesion may be limited to the microcarrier.

In addition, compounds promoting and inhibiting cell attachment can beused in combination. For example, the luminal surface of the BAO can betreated with compounds inhibiting cell attachment, and cell-carryingmicrospheres, or the matrix surrounding the cells (if used), may betreated with compounds promotimg cell attachment.

In another embodiment, the interior of the BAO may be altered byproviding an inert scaffold within the BAO prior to loading cells. Thisscaffold provides a structure for adhering and evenly distributing cellswithin the capsule. Compounds useful in the preparation of an inertscaffold include, poly(hydroxyethyl methacrylate) (“PHEMA”) andpoly(hydroxyethyl methacrylate-co-methyl methacrylate) (“PHEMA/MMA”).Furthermore, the scaffold may be derivatized with various chemicals orproteins, including those discussed supra, to further control growth anddifferentiation. According to this method, solutions of a suitablescaffold material are precipitated in the BAO for the desired scaffold.

Another embodiment contemplates culturing cells on a member which willserve as an internal support. The internal support may be made of anysubstantially biocompatible material such as titanium or a suitablepolymer. The support can be in the form of a strut or may be designed toalso function as a scaffold, by providing a large amount of surface areafor cell growth. One example of such a scaffold material is a non-wovenpolyester fabric (NWPF) (Reemay, Tenn.). There are numerous types ofNWPF, varying in tightness of weave and thickness of the sheet. Suchtechnique allows precise control over number of cells in a BAO, as wellas the ability to qualify the cells/scaffold prior to insertion in theBAO. Further, differentiation of cells cultured on such a material(external to the device) could be accomplished prior to insertion of thematerial into the device. Such a scaffold could be modified, forexample, with cell adhesion peptides, to induce cellulardifferentiation. Additionally, the material adds strength to the BAO.The fabrication of BAOs containing an inner support is described inco-pending application Ser. No. 08/105,728.

The BAOs useful in this invention typically have at least onesemipermeable outer surface membrane or jacket surrounding acell-containing core. The jacket permits the diffusion of nutrients,biologically active molecules and other selected products through theBAO. The BAO is biocompatible, and preferably immunoisolatory. The corecontains isolated cells, either suspended in a liquid medium orimmobilized within a hydrogel matrix.

It is to be understood that the foregoing methods and compositions forcontrolling the distribution of cells within a BAO are not exclusive. Itmay be desireable to use several of the methods and compositions incombination to achieve the desired growth control.

For example, it may be desirable to produce cells that have beengenetically modified to include a growth controlling gene according tothe methods of this invention, grow those cell on ECM microcarriers, andencapsulate the cell/microcarrier clusters in a BAO in which one or moregrowth surfaces have been modified to control cell distribution.

The encapsulating membrane of the BAO may be made of a material which isthe same as that of the core, or it may be made of a different material.In either case, a surrounding or peripheral membrane region of the BAOwhich is permselective and biocompatible will be formed. The membranemay also be constructed to be immunoisolatory, if desired.

The choice of materials used to construct the BAO is determined by anumber of factors and is described in detail in Dionne WO 92/19195.Briefly, various polymers and polymer blends can be used to manufacturethe capsule jacket. Polymeric membranes forming the BAO and the growthsurfaces therein may include polyacrylates (including acryliccopolymers), polyvinylidenes, polyvinyl chloride copolymers,polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulosenitrates, polysulfones, polyphosphazenes, polyacrylonitriles,poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymersand mixtures thereof.

BAOs may be formed by any suitable method known in the art. One suchmethod involves coextrusion of a polymeric casting solution and acoagulant which can include biological tissue fragments, organelles, orsuspensions of cells and/or other therapeutic agents, as described inDionne, WO 92/19195 and U.S. Pat. Nos. 5,158,881, 5,283,187 and5,284,761, incorporated herein by reference.

The jacket may have a single skin (Type 1, 2), or a double skin (Type4). A single-skinned hollow fiber may be produced by quenching only oneof the surfaces of the polymer solution as it is co-extruded. Adouble-skinned hollow fiber may be produced by quenching both surfacesof the polymer solution as it is co-extruded. Typically, a greaterpercentage of the outer surface of Type 1 hollow fibers is occupied bymacropores compared to Type 4 hollow fibers. Type 2 hollow fibers areintermediate.

Numerous capsule configurations, such as cylindrical, disk-shaped orspherical are possible.

The jacket of the BAO will have a pore size that determines the nominalmolecular weight cut off (nMWCO) of the permselective membrane.Molecules larger than the nMWCO are physically impeded from traversingthe membrane. Nominal molecular weight cut off is defined as 90%rejection under convective conditions. In situations where it isdesirable that the BAO is immunoisolatory, the membrane pore size ischosen to permit the particular factors being produced by the cells todiffuse out of the vehicle, but to exclude the entry of host immuneresponse factors into the BAO. Typically the nMWCO ranges between 50 and200 kD, preferably between 90 and 150 kD. The most suitable membranecomposition will also minimize reactivity between host immune effectormolecules known to be present at the selected implantation site, and theBAO's outer membrane components.

The core of the BAO is constructed to provide a suitable localenvironment for the particular cells isolated therein. The core cancomprise a liquid medium sufficient to maintain cell growth. Liquidcores are particularly suitable for maintaining transformed cell lineslike PC12 cells. Alternatively, the core can comprise a gel matrix. Thegel matrix may be composed of hydrogel (alginate, “Vitrogen™”, etc.) orextracellular matrix components. See, e.g., Dionne WO 92/19195.

Compositions that form hydrogels fall into three general classes. Thefirst class carries a net negative charge (e.g., alginate). The secondclass carries a net positive charge (e.g., collagen and laminin).Examples of commercially available extracellular matrix componentsinclude Matrigel™ and Vitrogen™. The third class is net neutral incharge (e.g., highly crosslinked polyethylene oxide, orpolyvinylalcohol).

Any suitable method of sealing the BAO may be used, including theemployment of polymer adhesives and/or crimping, knotting and heatsealing. These sealing techniques are known in the art. In addition, anysuitable “dry” sealing method can also be used. In such methods, asubstantially non-porous fitting is provided through which thecell-containing solution is introduced. Subsequent to filling, the BAOis sealed. Such a method is described in copending U.S. application Ser.No. 08/082,407, herein incorporated by reference.

One or more in vitro assays are preferably used to establishfunctionality of the BAO prior to implantation in vivo. Assays ordiagnostic tests well known in the art can be used for these purposes.See, e.g., Methods In Enzymology, Abelson [Ed], Academic Press, 1993.For example, an ELISA (enzyme-linked immunosorbent assay),chromatographic or enzymatic assay, or bioassay specific for thesecreted product can be used. If desired, secretory function of animplant can be monitored over time by collecting appropriate samples(e.g., serum) from the recipient and assaying them. If the recipient isa primate, microdialysis may be used.

The number of BAOs and BAO size should be sufficient to produce atherapeutic effect upon implantation is determined by the amount ofbiological activity required for the particular application. In the caseof secretory cells releasing therapeutic substances, standard dosageconsiderations and criteria known to the art are used to determine theamount of secretory substance required. Factors to be considered arediscussed in Dionne, WO 92/19195.

Implantation of the BAO is performed under sterile conditions.Generally, the BAO is implanted at a site in the host which will allowappropriate delivery of the secreted product or function to the host andof nutrients to the encapsulated cells or tissue, and will also allowaccess to the BAO for retrieval and/or replacement. The preferred hostis a primate, most preferably a human.

A number of different implantation sites are contemplated. Theseimplantation sites include the central nervous system, including thebrain, spinal cord, and aqueous and vitreous humors of the eye.Preferred sites in the brain include the striatum, the cerebral cortex,subthalamic nuclei and nucleus Basalis of Meynert. Other preferred sitesare the cerebrospinal fluid, most preferably the subarachnoid space andthe lateral ventricles. This invention also contemplates implantationinto the kidney subcapsular site, and intraperitoneal and subcutaneoussites, or any other therapeutically beneficial site.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of thisinvention in any manner.

EXAMPLES Example 1

Growth Control Using the Mx1 Promoter

The mouse Mx1 promoter was fused with the SV40 early region and thechimeric gene was used to generate transgenic mice. Because the Mx1promoter elements are induced in the presence of IFNα or IFNβ, oncogeneexpression in tissues or in cell cultures prepared from the transgenicanimals can be controlled.

Thus, conditionally-immortalized cell lines can be generated.

Production Of Transgenic Mice

The Mx1-Tag construct we used consisted of approximately 2 kb of the Mx1promoter (i.e., Xba1-EcoR1 fragment) fused to an intact SV40 earlyregion cDNA, which encodes both large T and small T antigens and isfused upstream of the mouse beta globin 3′ untranslated region andpoly-A signal (BamH1-Xbal fragment). The beta globin sequences wereincluded to provide splice sites and to enhance expression of the cDNAin transgenic animals. FIG. 1 shows the plasmid map of the Mx-1construct.

Transgenic mice containing the Mx1-Tag construct were produced by thestandard technique of pro-nuclear microinjection into single-cellfertilized mouse ova (Brinster et al., Proc. Natl. Acad. Sci. USA, 82,pp. 4438-4442 (1985)). Southern blot analysis of tissues from thefounder animals confirmed that intact copies of the transgene wereintegrated in the genome.

Offspring from these mice were confirmed as “DNA positive” using PCRamplimers that recognize sequences of the SV40 early region.

Conditionally-Immortalized Stem Cells

Striata were removed from E15 transgenic mouse embryos and DNA negativelittermates and plated in primary (individual) cell culture inEGF-containing neurospheres medium (per 100 mls: DDH₂O 50 ml,10×DMEM/F12 10 ml, 30% glucose 2.0 ml, NaHCO₃ 1.5 ml, 1M HEPES 0.5 ml,L-glutamine 1.0, 10×hormone mix 10 ml, DDH₂O 25 ml (to wash filter)).Neurospheres were prepared according to the method of Weiss, PCTCA92/00283, and Reynolds and Weiss, J. Neuroscience, 12, pp. 4565-74(1992). Cells were passaged seven times once a week and then dividedinto 2 groups: with and without exogenous interferon (IFN). Cells wereplaced in T25 flasks at a plating density of 500,000 cells/5 ml inEGF-containing neurosphere medium. 1000 units/ml IFN were added to ½ ofthe cells. Control neurospheres received no IFN. The cells wereincubated at 37° C., 5% CO₂ and were passaged weekly.

After 30 passages (23 with IFN), the cells were placed inserum-containing medium (DMEM, 5% fetal bovine serum, and 1×L-glutamine)with 1000 units of IFN at a cell density of 1.25 million cells in 15 ml.Fresh IFN was added every other day.

Seven days later, the medium was removed, the cells were washed withHanks' Balanced Salt Solution (HBSS), and the flask was lightlytrypsinized. The cells were resuspended in 10 ml of the serum-containingmedium, spun down at 1000 RPM for 2 minutes, and the medium wasaspirated off. The cells were then resuspended in 2 ml of serum mediumby triturating with a fire-polished pipet.

Approximately 25,000 cells were plated on poly-ornithine-treatedcoverslips in DMEM with 5% FBS. IFN was added to half of the coverslips(1000 units/ml) every other day. Cells were stained for SV40 T-antigen(Tag) and glial fibrillary acidic protein (GFAP), an intermediatefilament protein specifically expressed in astrocytes, at variousintervals, according to the following protocol.

Coverslips were immersed in 4% paraformaldehyde in 0.1M phosphatebuffered saline (PBS) for 20 mins. at room temperature, and then washedtwice for 5 mins. in PBS. Cells were permeabilized in 100% EtOH for 2min, and then washed again twice for 5 min. in 0.1 M PBS. Cells wereblocked with 5% NGS (normal goat serum) diluted in 0.1 M PBS for atleast 30 mins at room temperature. Primary antibodies were pooled anddiluted in 1% NGS for 2 hrs. and were applied to the coverslips at roomtemperature, as follows: anti-Tag (mouse monoclonal) was diluted 1:10,anti-GFAP (rabbit polyclonal) was diluted 1:500. The primary antibodieswere removed and the coverslips were then washed twice for 5 mins. withPBS.

Secondary antibodies were pooled and diluted in 1% NGS and were appliedto the coverslips for 30 min. at room temperature in the dark, asfollows: GAM-FITC (1:128); GAR-Texas Red (diluted 1:100). The secondaryantibodies were removed and the coverslips were washed twice for 5 mins.with PBS in the dark.

The coverslips were mounted with Citiflour™ (or other anti-fadentmounting media) onto slides and stored at 4° C. until viewing using afluorescent microscope equipped with rhodamine and fluorescein optics.

In this set of experiments, we set out to determine how quicklyT-antigen levels fall upon the removal of the interferon. In addition,we were interested to determine the effect of T-antigen level on cellproliferation and differentiation. Differentiation was assessed bymonitoring GFAP level. GFAP is an intermediate filament proteinspecifically expressed in mature astrocytes. The followingimmunofluorescence results were observed.

IFN (1000 units/ml) Control (No IFN) Day Tag GFAP Tag GFAP 1 +++ − +++ −4 +++ − + +/− 7 +++ − +/− +/− 10  +++ − − +

Thus, as shown by Tag and GFAP immunostaining, after a period of time inthe serum medium, the IFN-treated cells showed continued expression ofT-antigen, continued proliferation, and no evidence of GFAP expression,while the controls (no IFN) began to differentiate (upregulated GFAPexpression) and ceased dividing. This was confirmed by a visualinspection of the coverslips—there was a clear cut difference in cellnumbers by day 4. By day 10, the IFN-treated cells were much morenumerous than the controls.

The expression of the SV40 T-antigen in this construct is regulated in adose-dependent manner. In the cell lines we have produced, maximalT-antigen expression (measured by immunofluorescence) was observed at anIFN dose of 500-1000 units/ml. At 100 units/ml we observed minimal to noexpression. As would be expected, the rate of proliferation correlatedwith the IFN dose; there was little or no cell division at 100 units/mlof IFN.

In further studies with the above described Mx1 Tag EGF-responsiveneural stem cells, we have shown that proliferation and differentiationcan be controlled. A population of these stem cells were forced todifferentiate by removing EGF and adding FBS. With the addition of 1000Units/ml of alpha/beta IFN, clusters of flat, astrocyte-like cells beganto proliferate and eventually filled the culture dishes. We havecontinuously maintained these cells in IFN for over 70 passages and havemaintained a doubling rate of 24-36 hours over this period. When probedwith a panel of neural and glial-specific antibodies, these IF-treatedcells were virtually all nestin- and T-antigen-positive but were weaklyimmunoreacitve for glutamine synthetase and were GFAP-negative.

Upon removal of the IFN, these flat cells rapidly decreased their rateof division, lost T-antigen immunoreactivity and gradually increasedglutamine synthetase and GFAP immunoreactivity. These cells survive forseveral months in vitro and no proliferation is evident in the continuedabsence of IFN. Interestingly, T-antigen immunoreactivity and cellularproliferation can be re-induced with the addition of IFN. This providesa cell line with the capacity to proliferate or differentiate in acontrolled fashion.

Example 2

Cells prepared according to Example 1 are encapsulated and implanted ina human host.

Preparation Of PAN/PVC Fibers

Permselective hollow fibers are prepared via a dry jet-wet spinningtechnique (Cabasso, Hollow Fiber Membranes, vol. 12, Kirk-OthmerEncyclopedia of Chemical Technology, Wiley, New York, 3rd Ed., pp.492-517, 1980; Dionne, WO 92/19195; U.S. Pat. No. No. 5,158,881).Asymmetric hollow fibers are cast from solutions of 12.5%polyacrylonitrile polyvinyl chloride (PAN/PVC) copolymer in dimethylsulfoxide (w/w). Single-skinned or double-skinned fibers are produced.The fibers are collected into a non-solvent water bath, glycerinated,and dried. Cells are loaded at a density of 25,000 cells/μl into aPAN/PVC single-skinned hollow fiber and sealed by heat pinching.

Implantation into Host

The encapsulated cells are implanted into a human host. Implantationsites include the lateral ventricles and striatum of the brain.Procedures for implantation of BAOs into the brain are described inAebischer et al., WO 93/00127, incorporated herein by reference.

Example 3

Conditional Immortalization of Neonatal Astrocytes

A fragment containing the promoter elements of mouse mammary tumor virus(MMTV) is fused to the SV40 early region cDNA. E15 rat brain derivedneonatal astrocytes are transfected by electroporation and transformantsselected by assaying for proliferation. Dividing cells are removed,expanded and assayed for expression of large T-antigen, using anti-largeT antibodies. Transformed cells are encapsulated in BAOs and implantedin a host, substantially as described in Example 2. The BAOs are held invivo for one month. The BAOs are then retrieved and the celldistribution in the BAOs compared to cohorts held in vitro for the sametime period.

Example 4

Collagen-Reduced Proliferation and Ascorbate-Induced Differentiation OfSCT-1 Cells

SCT-1 cells were cloned from a sciatic nerve tumor from a Po-SV40transgenic mouse (Messing et al., J. Neuroscience, 14, pp. 3533-39(1994). These SCT-1 cells were immunoreactive for the Schwann cellmarkers S100 and Po, as well as for SV40 T-antigen.

SCT-1 cells were grown under three conditions: (1) on tissue cultureplastic without ascorbate, (2) on tissue culture plastic in the presenceof 50 μg/ml ascorbate to induce differentiation, and (3) suspended inType I collagen.

On a plastic substratum in the absence of ascorbate, most cellsdisplayed a fibroblast-like morphology. However, some bipolar cells werepresent. Cells doubled in 18-20 hours and displayed no contactinhibition.

SCT-1 cells grown in the presence of ascorbate demonstrated slowergrowth and a more robust staining for fibronectin and type IV collagen.Laminin immunoreactivity, on the other hand, was similar in control andascorbate-induced differentiated cultures.

SCT-1 cells suspended in Type I collagen exhibited a bipolar morphologyand a dramatic decrease in mitotic activity (i.e., doubling time was ≧30days).

Example 5

Inhibition of BHK Cell Proliferation by Ascorbate and TGF-β

BHK cells secreting CNTF were grown in DMEM (high glucose) medium.Treatment of subconfluent BHK cultures with TGF-β1 (2.5 ng/ml) andascorbate (100 μM) reduced mitosis. In addition, the cells appearedelongated, with some cells aligning. This data indicates TGF-β1 andascorbate inhibits proliferation and induces differentiation of BHKcells.

In further experiments, BHK cells secreting hNGF were treated with 2.5ng/ml TGFβ and 100 μM ascorbic acid prior to encapsulation in BAOs andimplantation. Non-treated cells served as controls. The specificvariables include: a) TGFβ/ascorbate, no Vitrogen™, b) TGFβ/ascorbate,Vitrogen™, c) no TGFβ/ascorbate, Vitrogen, and d) no TGFβ/ascorbate, noVitrogen™. In addition, several different polymers were used. Capsuleswere implanted into the striatum of adult rats. Rats were sacrificedafter 3 mos.

Example 6

Neural Stem Cells Proliferate in the Presence of EGF and Differentiatein its Absence

Neurospheres were prepared using the methods of Weiss et al., PCT/CA92/00283. Passage 68 neurospheres were collected and divided. Half ofthe neurospheres were triturated into a single cell suspension and halfremained as clusters. A single cell count was performed on a single cellsuspension and it was assumed that the clustered cells were of the sameconcentration. Single cells and clusters were suspended separately inequal amounts of Vitrogen™ and either neurosphere medium with 20 ng/mlEGF as controls, or PC-1 medium.

Cells were loaded at a density of 25,000 cells/μl into single-skinnedhollow fiber PAN/PVC BAOs, prepared substantially as described inExample 2, and then hub sealed. The BAOs were held in eitherneurosphere+EGF medium or in PC-1 medium (with no EGF).

The BAOs were sacrificed after 3 days and 7 days and were stained forglial fibrillary acidic protein (GFAP) by immunocytochemistry. GFAP isan intermediate filament protein specifically expressed in astrocytes.GFAP reactivity indicates that the neural stem cells have differentiatedinto astrocytes. The following results were observed:

Time (days) GFAP Reactivity Single cell, no EGF 3 Small % + for GFAPSingle cell, EGF 3 Negative Cell clusters, no EGF 3 Small % + for GFAPCell clusters, EGF 3 Negative Single cell, no EGF 7 Intense + for GFAPSingle cell, EGF 7 Negative Cell Clusters, no EGF 7 Intense + for GFAPCell Clusters, EGF 7 Negative

By day 7, the encapsulated neural stem cells had differentiated intoastrocytes in the absence of EGF.

Example 7

Effect of ECM on BHK Cells

Preparation of Acellular ECM

E15 rat meningeal cells obtained from 15 day old embryonic rats wereplated in multiwell plates and allowed to become confluent. The cellswere monolayer contracted after 2 weeks and were allowed to regrow.

Acellular ECM was extracted by treatment with 0.1 % Triton X-100detergent for 30 mins, and then treatment with 5 mM NH₄OH for 3 mins.

BHK-hNGF Cells

A BHK cell line secreting NGF was produced as follows. A 2.51 kbfragment containing approximately 37 bp of the 3′ end of the firstintron, the double ATG sequence believed to be the protein translationstart for pre-pro-NGF and the complete coding sequence and entire 3′untranslated region of the human NGF gene (Hoyle et al., Neuron, 10, pp.1019-34 (1993)) was subcloned into the DHFR-based pNUT expression vectorimmediately downstream from the mouse metallothionein-1 promotor (−650to +7) and the first intron of the rat insulin II gene (Baetge et al.,Proc. Natl. Acad. Sci., 83, pp. 5454-58 (1986)).

Baby hamster kidney (BHK) cells were transfected with the pNUT-βNGFconstruct using the calcium phosphate method. BHK cells were grown inDMEM containing 10% fetal bovine serum,1×penicillin/streptomycin/ampicillin B (0.8 g/l), and L-glutamine(GIBCO) in 5% CO₂ and at 37° C. Transfected BHK cells were selected inmedium containing 200 μM methotrexate (Sigma) for 3-4 weeks andresistant cells were maintained as a polyclonal population either withor without 200 μM methotrexate.

The transformed BHK-hNGF cells were plated at a density of 1.0×10⁴cells/well in the plates containing extracted ECM from meningeal cells.BHK-hNGF cells were also plated at the same density in control platesnot containing ECM. Cells were counted using a hemacytometer after 6DIV.

Cell counts for the control wells averaged 4.5×10⁶+/−4.5×10⁵ cells. Thecell counts for the extracted ECM plates averaged 9.9×10⁵+/−4.9×10⁵cells. These results show a 4.5 fold decrease in cell growth on thetreated plates.

Example 8

Adherence of Cells to Acellular ECM on an Inner Support

In further experiments, primary menigeal cells were seeded onto a TECOWpolyurethane fiber. Such fibers are useful as inner supports in BAOs.DMEM supplemented with 10% FBS was used as the culture medium. After 2weeks, the fibers were extracted with 0.1% Triton X-100 for 30 minutes,followed by 25 mM NH₄OH for 3 mins. Some fibers were immunostained withantifibronectin antibody to confirm the presence of acellular ECM on thefiber. Other fibers were used in a cell adhesion assay with BHK cells.

Example 9

BHK Cell Growth on Microcarriers Encapsulated in BAOs Modified withPEO-PDMS

Preparation of PEO-PDMS Derivatized BAOs

Single-skinned PAN/PVC hollow fiber BAOs were produced as described inExample 2. These BAOs had an ID of 642.6±36.7 μm, an OD of 787.8±32.2μm, a wall thickness of 67.8±16.2 μm, a BSA rejection coefficient of100%, and a hydraulic permeability of approximately 21.8 ml/min/m²/mmHg.

The PAN/PVC BAOs were derivatized with PEO-PDMS under sterileconditions. A 1% or 5% (v/v) solution of PEO-PDMS (Huls, PS073, MW=3126g/mole; 82% PEO by weight) was prepared by diluting 1 ml or 5 ml ofPEO-PDMS to 100 ml with deionized water. The solution was sterilefiltered (0.2 μm) prior to injection into a “wet” PAN/PVC membrane. Themembrane was heat pinched and immersed in an aqueous solution. Thefibers were rinsed with Hanks' Buffered Salt Solution after 72 hrs andprior to use with cells.

NGF-secreting BHK cells as described in Example 7, were loaded into thePEO-PDMS derivatized fibers as follows.

Loading and Sealing Procedure

Single cell suspensions of NGF-producing BHK cells grown to 90%confluency were rinsed with PBS (lacking calcium and magnesium),trypsinized for approximately 1 minute and pelleted by centrifugation at1000 rpm for 3 minutes. The cells were resuspended in medium to a finalcell concentration of 2×10⁷ cells/ml.

Cells were either loaded directly into the PEO-PDMS derivatized fibers,or mixed with a 0.15% Vitrogen® matrix solution or 0.5% agarosesolution, and then loaded. Approximately 2.5 microliters (ul) of cellsor cell/matrix slurry (10,000 cells/ul) were loaded into each fiberusing a 24-gauge beveled catheter tip and a Hamilton syringe.

Capsules were sealed by mounting a 1-1.1 cm length of dry hollow fiberonto a hub with a septal fixture at the proximal end which has loadingaccess for cells to be injected into the lumen of the device. Afterinfusing 2.5 μl of the cellular suspension, the septum was cracked offand the access port sealed using a light-cured acrylate (Luxtrak™ LCM24, ICI Resins US, Wilmington, Mass.) (“hub” sealed). The capsules weresubsequently “tethered” by placing a 1.5 cm 0.020″ silastic tube overthe acrylic hub.

The following BAOs were prepared in this manner:

1. control underivatized jacket, no matrix;

2. control underivatized jacket, Vitrogen® matrix;

3. control underivatized jacket, agarose matrix;

4. 1% PEO-PDMS derivatized jacket, no matrix;

5. 1% PEO-PDMS derivatized jacket, Vitrogen® matrix;

6. 1% PEO-PDMS derivatized jacket, agarose matrix;

7. 5% PEO-PDMS derivatized jacket, no matrix;

8. 5% PEO-PDMS derivatized jacket, Vitrogen® matrix;

9. 5% PEO-PDMS derivatized jacket, agarose matrix;

The BAOs were maintained at ambient O₂ for 4 days after encapsulation,and then maintained at low O₂ levels (50 mmHg) for the duration of thestudy. FIG. 2 shows NGF secretion (measured by ELISA) after 4, 11 and 25days.

The NGF release data indicates that the matrix alone has little effecton the output of the cells. However, in the presence of PEO-PDMS, theNGF release is substantially lower when used with agarose and without amatrix but not affected by when used with Vitrogen™. In addition, thepercent of PEO-PDMS used in the modification apparently had littleeffect on NGF release. From the histology data, the BHK cellsencapsulated with agarose had an elongated morphology and lined thewalls of the device; however, very few cells were viable within theagarose itself. The BHK cells loaded with agarose in PEO-PDMS-modifiedfibers also lined the inner luminal surface of the capsule but had around morphology. There were fewer cells in the PEO-PDMS-PAN/PVCmodified fibers than there were in the unmodified fibers with agarose,indicating that cell growth was controlled. The cells in Vitrogen™loaded devices were not affected by the fiber modification neither werethose encapsulated without a matrix.

BHK cells in unmodified fibers with a Vitrogen™ matrix were welldistributed with approximately 75% viability. There was some cellnecrosis in the center of the device. PEO-PDMS modification did notaffect cell distribution, viability or morphology. With agarose as thematrix, cell distribution was excellent with cell viabilityapproximating 90%. The cell morphology of BHK cells was affected byPEO-PDMS derivatization of the membrane (1% and 5%) when an agarosematrix was used. The cells were elongated in unmodified P(AN/VC) andmore rounded in modified P(AN/VC). Cells were not located in the agarosematrix, but in a space between the fiber and agarose “rod”. Without amatrix, the cell distribution is less satisfactory as cells have formedlarge clusters and the viability is lower (approximately) 60%.

Example 10

BHK Cell Growth on CultiSphers™

NGF-secreting BHK cells as described in Example 7 were grown on collagencoated CultiSphers™. CultiSphers™ (1 g) were rehydrated in 50 ml of PBS(CMF). 15×10⁶ cells were suspended in 1 ml of rehydrated CultiSphers™.The cell/CultiSphers™ suspension was loaded directly into single-skinnedPAN/PVC hollow fibers, or mixed in a 1:1 ratio with 1% agarose, and thenloaded into single-skinned PAN/PVC hollow fibers. The fibers wereprepared substantially as described in Example 2, and loaded and sealedsubstantially as described in Example 9.

The encapsulated cells were tested for NGF secretion by ELISA at 2, 15,and 56 days. The medium was replenished 3 times/week. FIG. 3 shows theresults. The NGF release data indicate that BHK cells can grow onCultiSphers™ microcarriers when encapsulated in BAOs (FIG. 3, legend:n-mat-008, 0709-n-m). Further, the NGF release data indicate that BHKcell/CultiSphers™ can be further suspended in an agarose matrix, withlittle or no effect on NGF secretion (FIG. 3, legend: agaro-008,agaro-0709).

Example 11

Use of a Peptide Derivative to Control Cell Number and Cell Distribution

In this example, the luminal surface of the BAO was modified withPEO-PDMS, poly(d-lysine), or PepTite 2000™, a commercially availablecell adhesion protein.

In this study baby hamster kidney (BHK) cells were used because they areanchorage-dependent cells and have been shown previously to adhere tothe hollow fiber membrane.

Fibers

Single-skinned PAN/PVC BAOs were produced substantially as described inExample 2. The fiber dimensions were 625 μm ID, 50 μm wall thickness.These fibers were sterilized by immersion in 70% ethanol overnight andthen rinsed repeatedly with HBSS.

Derivatization

1. PDMS-PEO: BAOs were derivatized with PDMS-PEO as follows. A 1% (v/v)solution of PEO-PDMS (purchased from Huls, PS073, Mw=3126 g/mole; 82%PEO by weight) was prepared by diluting 1 ml of PEO-PDMS to 100 ml withdeionized water. The solution was sterile filtered (0.2μm) prior toinjection into a sterile membrane. The membrane was immersed in a 1%PEO-PDMS aqueous solution for 24 h at room temperature. The fibers wererinsed with water (3 times) and then HBSS prior to injection of cells.

2. PdL: BAOs were derivatized with poly(d-lysine) as follows. Fiberswere immersed in an aqueous solution of 67,000 molecular weightpoly(d-lysine) at 2 mg/ml for 24 h at room temperature. The fibers wererinsed 3 times with water and then 3 times with HBSS prior to injectionof cells.

3. PopTite 2000™: BAOs were derivatized with PepTite 2000™ as follows.Fibers were immersed in a PBS solution of 100 mg/ml of PepTite 2000™previously dissolved in ethanol. The fibers were immersed in thissolution for 24 h at room temperature and then rinsed 3 times with PBSprior to injection of cells.

4. PAN/PVC: Control fibers were immersed in HBSS for 24 h at roomtemperature prior to injection of cells.

Calls

BHK cells were loaded into the derivatized fibers at a concentration of5000 cells/μl. The fibers were sealed and placed in screw-cap tubescontaining serum-free medium (PC1 medium) and then placed on a rotatingdrum for up to two weeks in an incubator set at 37° C. The drum speedwas 2rpm. At the appropriate time the fibers were fixed in 4%paraformaldehyde, dehydrated in graded ethanol and stained withhematoxylin and eosin (H&E) for histological analysis of celldistribution with osmium tetraoxide.

PAN/PVC-derivatized membranes showed a good distribution of cells whenderivatized with poly(d-lysine and a more even distribution of cellswhen derivatized with PepTite™ 2000, as determined by osmium tetroxidestaining.

For PAN/PVC membranes, PepTite 2000™ modifications were attempted in twoways. First, the inner luminal surface of the membranes was modifiedonly and second, both the inner luminal surface and the outer surfacewere treated. Empty BAOs (i.e. free of cells) were analyzed for totalamino acids, to determine the binding of poly(d-lysine) or PepTite2000™. The total amino acid bound to control, unmodified membranes wasapproximately 0.2 μg/BAO. The total amino acid bound topoly(d-lysine)-modified membranes was approximately 0.8 μg/BAO formodified inner luminal surface membranes, and approximately 2.6 μg/BAOfor membranes where both the inner luminal surface and outer surface hadbeen modified. Similar BAOs loaded with BHK cells were maintained for 14days, and then examined histologically. In control unmodified BAOs,cells were unevenly located in large clusters over the entire length ofthe fiber. In contrast, in both types of modified fibers, there was aneven distribution of cells along the luminal surface of the membrane.

These results suggest that poly(d-lysine) and PepTite 2000™ areeffective in promoting cell attachment to the BAO luminal surface, andthus are effective in controlling cell distribution within the BAO.

Example 12

Use of ECM Molecules to Control Growth of Neurosperes

Passage 71 mouse neurospheres were prepared substantially as inExample 1. Multi-well dishes were precoated with 0.5% agarose(Sea-Prep™) to keep the neurospheres from attaching to the plasticdishes. Cells were plated at a density of approximately 50,000 cells perwell into the designated matrices for the experiment. Three wells wereused for each matrix condition; two of the wells contained PC-1 medium(control) and one contained neurospheres+EGF medium(EGF).

A dermal-derived Type 1 collagen (Zydast™; (Collagen Biomedical, PaloAlto)), a tendon-derived Type 1 collagen (Organogenesis™), a Type 1collagen (Vitrogen™, Celtrix, Santa Clara), and agarose were evaluatedfor effectiveness in controlling cell growth, alone, or in combinationwith laminin or PepTite 2000™, or both.

At 4 days and 14 days cells were assayed by staining with fluoresceindiacetate/propidium iodide (FDA/PI), and were evaluated for cellviability, growth, and differentiation. Cells exposed to a combinationof the Organogenesis™ collagen, Peptite 2000™ and laminin showed thehighest amount of differentiation, with about 90% of the cells havingundergone differentiation. About 80% of cells exposed to a combinationof agarose, Peptite 2000™ and laminin had differentiated.

Example 13

Use of an Inert Scaffold to Control BHK Cell Number and CellDistribution in a BAO

Two types of PAN/PVC fibers (substantially as described in Example 2)were used: a single-skinned fiber having the permselective membrane onthe outer surface, and a single-skinned fiber having the permselectivemembrane on the inner surface.

First, PAN/PVC fibers were deglycerinized and sterilized by immersion in70% sterile filtered ethanol overnight. The fibers were then rinsed withsterile water three times over the course of about 1 to 2 hours.

Next, a 15% concentration poly(hydroxyethyl methacrylate) (“PHEMA”)scaffold matrix was prepared by dissolving 1.5 g PHEMA in 10 ml of 95%ethanol (190 proof, Quantum). In addition, a 10% concentrationpoly(hydroxyethyl methacrylate-co-methyl methacrylate) (“PHEMA/MMA”)scaffold matrix was made by dissolving 1.0 g of PHEMA/MMA in 10 ml of95% ethanol. To dissolve the polymers more easily, the solution wasstirred and heated.

The PHEMA or PHEMA/MMA solutions were loaded with a syringe into thePAN/PVC fibers, which were then immersed in sterile water. The loadedfibers were left in water for more than 1 hour to ensure precipitationof the scaffolds and diffusion of ethanol out of the core. The ends ofthe fibers were cut off because they were often clogged with eitherPHEMA or PHEMA/MMA. The fibers were transferred to Petri dishescontaining sterile HBSS. BAOs loaded with PHEMA, PHEMA/MMA and controlBAOs were prepared in this manner.

NGF-secreting BHK cells (described in Example 7) were grown in 10% DMEMwith glutamine and antibiotics added. The cells were gently pulled offthe flasks with 0.25% trypsin, washed and resuspended in PC1 media to adensity of 1×10⁷ cells/ml.

The BHK-NGF cells were loaded into the fibers at a density of 10,000cells/μl using a 22 gauge Teflon catheter. BAOs were sealed by heatpinching.

Five BAOs of each type were prepared. Four were placed in a 24 wellplate with 1 ml of PC-1 media. The fifth was placed in approximately 3-4ml of PC-1 media in a vertical tube. After 24 hours, the BAOs placed inthe vertical tube were cut open along the lumen (longitudinalcross-section) and analyzed after 24 hours by staining with fluoresceindiacetate/propidium iodide (FDA/PI) for cell distribution within thefibers. When viewed under a fluorescent microscope, FDA stains viablecells green and PI stains non-viable cells red.

The remaining BAOs were cultured for 2 weeks. The BAOs were maintainedat ambient O₂ for 4 days after encapsulation, and then maintained at lowO₂ levels (50 mmHg) for the duration of the study.

The functionality of BHK-NGF cells was tested by measuring NGF secretion(by ELISA) after 4, 7 and 14 days. The cells PHEMA or PHEMA/MMAscaffold-containing BAOs continued to secrete NGF over the duration ofthe study. Both the histology and NGF-release data indicate that PHEMAand PHEMA-MMA scaffolds allow maintenance of functionally-active viablecells distributed along the BAO. The results with 10% PHEMA-MMAscaffolds were the best.

Example 14

Use of an Inert Scaffold to Control PC12A Cell Number and CellDistribution in a BAO

The effectiveness of PHEMA and PHEMA/MMA inert scaffolds were evaluatedfor effectiveness in controlling the distribution of PC12 in BAOs.

Single-skinned fibers were prepared substantially as described inExample 2. These fibers typically had the following characteristics: 642μm ID, 787 μm OD, wall thickness 68 μm, rejection coefficient 100%(BSA), hydraulic permeability 22 ml/min/m2/mm Hg.

Inert scaffolds of PHEMA and PHEMA/MMA were prepared in these fibers,substantially as described in Example 13.

PC12A cells (1×10⁷ cells/ml) in HL-1 medium were injected into thelumens of the fibers, and the fibers heat sealed to produce BAOsapproximately 1 cm long. The devices were held at 37° C. at ambientpressures in HL-1 media. To assess functionality of the encapsulatedcells, the BAOs were tested for basal and K⁺-evoked catecholaminerelease at 1, 14 and 28 days. The results are shown in FIGS. 4A and 5A(basal release) and FIGS. 4B and 5B (K⁺-evoked release). These resultsshow that PC12 cells encapsulated in BAOs having inert PHEMA andPHEMA/MMA scaffolds retain their functionality, as measured bycatecholamine release.

Cell distribution in the BAOs was evaluated after 5 hours and 4 days byvertically cutting the fibers in half, and staining the cells withFDA/PI. These results indicated that PHEMA and PHEMA/MMA scaffolds arenontoxic and support cell viability and functionality of PC12 cells.

Example 15

Use of an NWPF to Promote Cell Adhesion and Differentiation in a BAO.

Six types of NWPF (Reemay, Tenn.) were tried: #2470, #2295, #2024,#2055, #2033, #2250 (Reemay #s). The fabric received was in flatsheetform: discs were punched out to fit into 24 well plates. The NWPF discswere immersed in 1% sodium dodecyl sulphate (SDS), w/v for 6 h and thenrinsed with water (3 times). The discs were then immersed in 1% sulfuricacid (v/v in H₂O) for 13 h (overnight) and then rinsed 3 times withwater. The discs dried on a paper towel and then sterilized byautoclaving.

The discs were cultured with 3 cell types to test for cell adhesion:BHK, AT-3, and TSA cells. Approximately 100,000 cells were added to a 24well plate containing one of the above 6 NWPF discs in PC1 media. Aserum-free medium was used to test for cell adhesion without theinference of serum (except for TSA cells). After 4 days, the BHK andAT-3 cells were examined for adhesion by PDA/PI. The cells had anelongated morphology and appeared to adhere on Reemay #2250, and 2055.At 10 days, BHK were growing best on #2250. AT-3 cells best adhered to2024 and 2295. AT-3 cells grew best on 2024 at 10 days. TSA cells (in10% FCS) after 1 day had an elongated morphology when grown on #2250,#2055, and grew best on #2024. At 7 days, TSA cells were growing best on#2055.

Example 16

SV40/DβH-NGF Cells on Microcarriers Suspended in Matrix Material

Regulatory elements of the dopamine β-hydroxylase (DβH) gene (Hoyle etal., J. Neurosci., 14, pp. 2455-63 (1994)) were utilized to direct thecoexpression of the SV40 T-antigen (tsa58) (DβH-SV) and human growthfactor (DβH-hNGF) in transgenic mice. Coexpression of the chimeric genesresulted in neoplasms in the adrenal medulla and noradrenergicsympathetic ganglia. A tumor of the celiac region from one of these micewas dissected and the tumor tissue was mechanically dissociated andplaced in cell culture (DMEM, 10% FBS, 37° C., 5% CO₂). Two distinctcell types, large flat fibroblast-like cells and small phase-brightcells having extensive neurite processes, were present from the initialculture period. The small cells exhibited features of catecholaminergicneuron including immunoreactivity for neurofilament-L and -M andtyrosine hydroxylase. Immunoreactivity for the SV40 T-antigen was alsopresent in these cells, in contrast to the fibroblast-like cells, whichwere negative for these markers. The cells were passaged weekly.

Cells were grown on an CultiSphers™ as described in Example 10, and weresuspended in either an alginate (1.5%) or agarose (1%) matrix. In thecase of the alginate matrix, the alginate was cross-linked by immersingthe devices in a 1% aqueous calcium chloride solution for 5 minutesafter encapsulation. The cells/Cultisphers™/matrix were loaded intoPAN/PVC hollow fibers as described in Example 10.

The cell-loaded BAOs were maintained in serum-free medium conditions. Atselected time intervals, devices were washed prior to 30 minuteincubations in HBSS. The basal medium was collected and assayed byHPLC-ED for L-dopa. The devices continued to secrete L-dopa at 80 daysin vitro.

Example 17

Genetically Modified Myoblasts Secrete NGF After Differentiation

Mouse C₂C₁₂ myoblast cells have the advantage of being rapidly dividingcells, can be grown in large quantity in vitro, transferred to expressproteins and selected clones can be isolated. Mouse C₂C₁₂ cells can bedifferentiated into a post-mitotic state upon exposure to low serumcontaining medium. These cells are thus advantageous for encapsulationin comparison to dividing cells whose proliferation cannot becontrolled—the latter cells continue to divide until they fill thecapsule and an accumulation of debris is observed after several months.

We tested the ability of a transfected C₂C₁₂ myoblast line to continuesecreting hNGF after fusion into myotubes has taken place.

C₂C₁₂ myoblast cells (ATCC) were transfected with a hNGF gene, using theLipofectamine reagent following the manufacturer's protocol (Gibco).Cells were selected in 1 mg G418 for 2 weeks and then tested for NGFoutput. Cells were plated at about 260 cells/cm2 in T75 flasks and 24well plates with or without cover slips. Cells were fed twice a weekwith DMEM and 10% FBS. Cells were harvested at 1, 5, 8, and 13 days, atwhich time NGF secretion was measured. The results are shown in Table 2.

TABLE 2 Time Course of C₂C₁₂ screening For NGF Secretion cell line timein % % secretion culture confluency fusion NGF parent day 1 25 0 nt3/23/95 +NGF day 1 25 0 nt 3/23/95 parent day 5 40 0 nt +NGF day 5 30 0nt parent day 8 98 5 *** +NGF day 8 90 1 0.0018 parent day 13 1000  80 ND +NGF day 13 100  50  0.014  % fusion = % myoblast cells forming intomyotubes “+NGF” indicates C₂C₁₂ cells transfected with hNGF gene“parent” indicates untransfected C₂C₁₂ cells NGF secretion measured inpg/ml/cell/24 hr. nt = not tested ND = not detected *Day 8 Cells haveincreased in size, preparing for fusion. *Day 8 More fusion in theculture dishes than in the T flasks (Flow cytometry done on flasks)

These results suggest that transfected myoblasts continue to secrete thedesired heterologous product, i.e., NGF, after terminal differentiationinto myotubes.

Example 18

Genetically Modified Myoblasts Secrete CNTF After Differentiation

We transfected mouse C₂C₁₂ myoblasts with the pNUT expression vector(Baetge et al., Proc. Natl. Acad. Sci. USA, 83, pp. 5454-58 (1986)containing the human CNTF gene. The level of expression of the hCNTFgene and the bioactivity of the factor were analyzed by Northern blot,Elisa assay, and ChAT activity on embryonic spinal cord motoneuroncultures. One C₂C₁₂ clone was found to secrete approximately 0.2 gCNTF/10 cells/day. The rate of secretion of hCNTF was not altered upondifferentiation of C₂C₁₂ myoblasts. Finally, C₂C₂-hCNTF could rescuemotoneurons from axotomy-induced cell death. Morphological study of thefacial nuclei of newborn rates, 1 week after axotomy, indicated thatonly 13.4% of the facial motoneurons were retained in control animalswhereas a continuous release of hCNTF resulted in 22.7% survival of themotoneurons.

We claim:
 1. An isolated cell transformed with a recombinant DNAmolecule comprising: a) a proliferation-promoting gene for inducing celldivision when expressed, b) an Mx-1 promoter operably linked to theproliferation-promoting gene, wherein said isolated cell is induced toproliferate by exposure to an amount of interferon sufficient to resultin expression of the proliferation-promoting gene.
 2. The cell of claim1 wherein the proliferation-promoting gene is SV 40 large T antigen. 3.The cell of claim 1 wherein the cell secretes a biologically activemolecule.
 4. The cell of claim 3 wherein the cell is geneticallytransformed with an expression vector containing a gene that encodes thebiologically active molecule.
 5. The cell of claims 3 or 4 wherein thebiologically active molecule is selected from the group consisting ofneurotransmitters, hormones, cytokines, growth factors, trophic factors,lymphokines, angiogenesis factors, antibodies, blood coagulationfactors, and enzymes.
 6. The cell of claim 1 wherein the cell isobtained from a neural stem cell.
 7. A transgenic mouse, whose genomecomprises a recombinant DNA molecule comprising: a) aproliferation-promoting gene for inducing gene expression whenexpressed, b) an Mx-1 promoter operably linked to theproliferation-promoting gene, wherein said transgenic mouse containscells which are induced to proliferate by exposure to an amount ofinterferon sufficient to result in expression of theproliferation-promoting gene.
 8. The transgenic mouse of claim 7,wherein the proliferation-promoting gene is SV-40 large T antigen. 9.Progeny of the transgenic mouse of claim
 7. 10. A cell isolated from thetransgenic mouse of either claim 7 or
 9. 11. The cell of claim 10,wherein the cell is a neural stem cell.
 12. A method of generating aconditionally immortalized cell, comprising the steps of: a)transforming an isolated cell with a recombinant DNA molecule comprisinga proliferation-promoting gene for inducing cell division when expressedand an Mx-1 promoter operably linked to the proliferation-promotinggene, such that said transformed cell is induced to proliferate byexposure to an amount of interferon sufficient to result in expressionof the proliferation-promoting gene; b) and culturing said transformedisolated cell.